BG107218A - Use of il-18 inhibitors for the treatment and/or prevention of atherosclerosis - Google Patents

Abstract

The invention relates to the use of an IL-18 inhibitor for the manufacture of a medicament for the treatment and/or prevention of atherosclerosis. 48 claims, 9 figures

Description

USE OF IL-18 INHIBITORS FOR THE TREATMENT AND / OR PREVENTION OF ATHEROSCLEROSIS

TECHNICAL FIELD

The present invention is in the field of vascular diseases. In particular, the invention relates to the use of IL-18 inhibitors for the treatment and / or prevention of atherosclerosis.

BACKGROUND OF THE INVENTION

Atherosclerosis is a widespread and very important vascular disease, but many other vascular disorders are known. Basically, atherosclerosis affects the large arteries and those of medium size and lesions and includes fatty seals, phobrolytic plaques and lesions with complications.

Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by the progressive accumulation of lipids such as cholesterol, cells such as macrophages, T lymphocytes or smooth muscle cells and the extracellular matrix (1). Large deposits are called atheromas or plaques that often contain calcium. The adipose tissue can destroy the wall of the artery, reducing the elasticity of the artery and thus obstructing blood flow. Possibly, blood clots may form around the deposited plaques, which further impede blood flow, which can lead to a general blockage of the blood vessel. Typically, atherosclerosis is associated with increased levels of LDL-cholesterol, Lp (a) fibrinogen and factor VII, as well as decreased levels of HDL-cholesterol. Risk factors include increased age, male, smoking, diabetes, obesity, high blood cholesterol, foods rich in fat and humans, individually or family-laden with heart disease. It most commonly causes organ ischemia, such as myocardial infarction.

Atheroma is a widespread damage to the arteries, which may be further complicated by thrombo-embolism. Atheromatous plaques often constrict the lumen of the arteries, causing ischemia, and sometimes atrophy of tissues in the hypoperfusion region. Serious consequences include the symptom of angina due to myocardial ischemia, heart failure due to ischemia or non-ischemic cases, and hypertension due to renal artery narrowing and renal hypoperfusion, whose physiological response is increased secretion .

Sometimes atherosclerosis and arteriosclerosis are referred to as separate pathological conditions and in this case, atherosclerosis is defined as involving hardening (sclerosis) or loss of elasticity of the arteries due to atheroma, whereas arteriosclerosis is hardening or loss of hardness for any other reason.

Complications or sequelae of atherosclerosis include coronary artery disease (coronary artery atherosclerosis), insufficient blood supply due to obstruction (ischemia / angina,) acute MI (myocardial infarction, heart attack), transient ischemic attack, or stroke blood vessels, muscles or body organs.

Aneurysms, which are permanent, abnormal dilations of blood vessels, are also common consequences of atherosclerosis. Atherosclerotic abdominal aortic aneurysms usually develop in adult patients. They may burst into the retroperitoneal space. In atherosclerotic aneurysms there is usually a pronounced loss of elastic tissue and fibrosis of the media, mainly due to ischemia of the aortic muscle muscle, followed by the release of macrophage enzymes leading to fragmentation of the elastic fibers.

Drugs that are recommended for the treatment or prevention of atherosclerosis include lowering fat / cholesterol in the blood. In particular, the therapy for lowering LDL-cholesterol is widely used. Statins, which are specific inhibitors of HMG CoA reductase, are currently most widely used. In addition, other fat-lowering components include drugs such as cholestyramine, colestipol, nicotinic acid, gemfibrozil, probucol, lovastatin, and the like.

Another approach is to reduce the risk of blood clots in atheromatous lesions. Aspirin, which is considered to be a specific inhibitor of thromboxane A2 mediated platelet aggregation or anticoagulant, may be used to reduce the risk of thrombosis.

Subcutaneous balloon angioplasty uses a balloon-tip catheter to smooth out plaques and increase blood flow through obstructed areas. The technique is similar to that used to open the arteries of the heart, but it can also be applied to many other arteries in the body. Coronary artery stenosis bypass is a part of a soap vein, sutured with the proximal aorta or by dissection of the internal mammary artery from the chest wall and joining the distal end with an artery from the anterior surface of the heart.

Surgical debridement (endarterectomy) may only be recommended in some cases (for example: endarterectomy of the carotid artery).

However, the main recommendation is to treat or control risk factors such as maintaining low fat, low cholesterol and low salt foods and to follow health recommendations for the treatment and control of hypertension, diabetes and other diseases, body weight and smoking cessation, as well as periodic examination to determine the normal state and functioning of the heart.

The inflammatory process is involved in all the different stages of atherosclerosis (1). The first controllable stage of atherosclerosis is thought to be endothelial activation by various factors, including weaker cell contact, modified lipoproteins, and pro-inflammatory cytokines (1). Many studies have recently shown that interactions between vascular cells and those at the site of inflammation are critical for atherosclerosis (1). In particular, suppression of certain pre-inflammatory pathways reduces the development of atherosclerosis (1).

Inflammation plays a major role in the destruction of atherosclerotic plaques and thrombosis (2 - 5) and therefore influences cases of acute ischemic syndromes and associated mortality (6). In fact, the severe clinical manifestations of atherosclerosis, including heart, brain, and other organs affected by atherosclerosis, are mainly due to obstruction of the vascular lumen by thrombi resulting from contact with the destroyed atherosclerotic plaque (3, 4).

Pathological studies have shown that vulnerable or unstable plaques, i. plaques prone to tearing or already tearing differ greatly in cellular composition and matrix composition compared to stable non-tear-resistant plaques (7). Vulnerable plaques are abundant in inflammatory cells (macrophages and T lymphocytes) and contain a thrombogenic lipid core and are characterized by a fine fibrotic coating with significant loss of extracellular matrix (7).

Reduced collagen synthesis mediated by the proinflammatory cytokine IFNγ and increased activity of macrophage-derived matrix degrading metalloproteinases are responsible for the thinning and brittleness of the fibrotic coating (7). The rupture of the fragile fibrous coating strongly exposes the thrombogenic lipid core in the circulating blood and leads to the formation of thrombosis (1, 7). Therefore, cell density due to inflammation in an atherosclerotic lesion is considered to be a good indication of its instability.

The clinical prognosis for a patient with atherosclerosis depends only partially on the size of the lesion (19; 20). Today it is widely accepted that quality (composition of the plaque) rather than the size of the lesion may even be a better indicator of the occurrence of ischemic cases. In fact, severe clinical manifestations of atherosclerosis (heart and brain infarctions) are mainly due to obstruction of the vascular lumen due to thrombus formation at the contact of the destroyed atherosclerotic plaque (19). Pathological studies have shown that vulnerable or unstable plaques that are prone to tearing or torn are rich in inflammatory cells and show significant loss of smooth muscle cells and collagen content (20, 21). Furthermore, such plaques show a significant increase in apoptotic cell death leading to the formation of a highly thrombogenic lipid core (13,22).

Προ-inflammatory cytokines are involved in inflammation.

The cytokine interleukin 18 (IL-18) was originally described as ySinterferon (IFN-γ) inducing factor (8). It is an early signal in the development of T-lymphocyte helper cell type 1 (Th1) responses. IL-18 works in conjunction with IL-12, IL-2, antigens, mitogens, and probably other factors to induce IFN-γ production. IL-18 also increases the production of GM-CSF and IL-2, potentiates anti-CD3 induced T cell proliferation and enhances Fas-mediated destruction of natural killer cells. Mature IL-18 is derived from its precursor by the ILΙβ conversion enzyme (ICE, caspase-1). The IL-18 receptor consists of at least two components that cooperate in ligand binding. High- and low-affinity binding sites for IL-18 are C found in murine IL-12 stimulated T cells (9), suggesting a multi-stranded receptor complex. So far, two receptor subunits have been identified, both belonging to the IL-1 receptor family (10).

IL-18 signal transduction involves activation of NF-κB (I) ·

Recently, soluble protein has been isolated from human urine, which has a strong affinity for IL-18, and human and mouse cDNAs have been cloned as well as the human gene (12; WO 99/09063). The protein is referred to as the IL-18 binding protein (IL-18BP).

IL18BP is not an extracellular domain of one of the known IL18 receptors but a secreted, naturally circulating protein. It belongs to a new family of secreted proteins, further comprising several Poxvirus-encoded proteins (12). IL18BP is continuously expressed in the spleen (12). The urine protein, as well as the recombinant IL18BP, bind specifically to high affinity IL-18 and modulate the biological affinity of IL-18.

The IL18BP gene is located in human chromosome 1 lql3 and no exon encoding a transmembrane domain is found in the 8.3 kb genomic sequence. Four splice variants or isoforms of IL18BP have been identified in humans and are designated IL18BP a, b, c and d, all having the same N-terminus and differing at their C-ends (12).

Four human and two mouse IL-18BP isoforms derived from mRNA splicing and found in various cDNA libraries have been expressed, purified and evaluated for binding and neutralizing the biological activities of IL-18 (23). The human IL-18BP isoform a (IL-18BPa) exhibits the highest affinity for IL-18 with a fast on and off rate and a dissociation constant (K (d)) of 399 pM. IL-18BP shares the Ig domain of IL-18BP a, with the exception of the C-terminal 29 amino acids; K (d) of IL-18BP c was 10-fold lower (2.94 nM). Nevertheless, IL-18BP a and IL-18BP with neutralize IL-18> 95% with a molar excess of both. The isoforms of IL-18BP b and IL-18BP d do not have a complete Ig domain and are not capable of binding or neutralizing IL-18. Mouse IL-18BP c and IL-18BP d isoforms have the identical Ig domain and also neutralize> 95% murine IL-18 with a molar excess of both. However, murine IL-18BP d, which has a common C-terminal motif with human IL-18BP?, Also neutralizes human IL-18. Molecular modeling identifies large mixed electrostatic and hydrophobic binding sites in the Ig domain of IL18BP, which may explain its strong affinity for ligand binding (23).

SUMMARY OF THE INVENTION

The invention is based on the finding that the IL-18 inhibitor has a distinct beneficial effect on plaque development, plaque progression and their stability in a murine model of atherosclerosis. The IL-18 inhibitor not only prevents lesion formation in the thoracic aorta, but also induces a switch to a stable plaque phenotype in already established atherosclerotic plaques.

The invention therefore relates to the use of an IL-18 inhibitor for the manufacture of a medicament for the prevention and / or treatment of atherosclerosis. In addition, the invention relates to methods of treatment, by gene therapy approach, for the treatment and / or prevention of atherosclerosis.

BRIEF DESCRIPTION OF THE GRAPHS

Figure 1 is a histogram showing the survival rate of human umbilical vein endothelial cells after incubation with oxidized lipoproteins alone, or incubated with a combination of oxidized lipoproteins and IL-18 antibody or IL 18BP respectively.

Figure 2 shows a Western blot performed with protein extracts from atherosclerotic arteries compared to control arteries. Antibodies against IL-18BP (hIL18BP), the IL-18 receptor α subunit (hIL18Ra), IL-18 (hIL18) and Caspase-1 (Caspase-1 pJ) were used in Western blots.

Figure 3 shows an ethidium bromide agarose gel showing the result of RT-PCR analysis for IL-18 and IL-18BP mRNA in atherosclerotic plaque cells.

Figure 4 Typical RT-PCR results for IL-18BP and IL-18 in atherosclerotic plaques compared with ha-actin (control) expression in symptomatic and asymptomatic plaques.

Figure 5 shows a map of the expression vector used for intramuscular electrotransfer in mice.

Figure 6 is a histogram showing the stained lipid region in atherosclerotic arteries. Computer generated image for quantitative analysis of lipid deposition. Data represent mean values is s.e.m. (η = 19 for empty plasmid, η = 14 for IL-18BP plasmid). The four stars indicate P <0.0001.

Figure 7 is a histogram showing a lesion of the aortic sinus region after IL-1SBP treatment compared to the control (empty plasmid). Computer generated image for quantitative analysis of the damaged area. Data represent mean values with s.e.m. (η = 19 for empty plasmid, η = 14 for IL-18BP plasmid). The two stars indicate P <0.01.

Figure 8 shows the effect of IL-18BP treatment on the content of damaged inflammatory cells. A computer-generated image for quantitative analysis is used to determine the percentage of macrophage-positive areas (black stripes) and the number of infiltrated T lymphocytes per mm

(gray lines) in aortic sinus lesions from control (n = 12 for macrophage staining, η = 15 for T lymphocyte staining) or IL-18BP treated mice (n = 13 for macrophages, η = 12 for T lymphocytes). Data represent mean values is s.e.m. The three stars indicate P <0.005; and the four stars indicate P <0.0001.

Figure 9 shows the effect of IL-18BP treatment on smooth muscle cell lesion and collagen content. A computer-generated image for quantitative analysis was used to determine the percentage of the positive region of smooth muscle cell (black lines) and collagen accumulation (gray lines) in aortic sinus lesions from control (n = 6 for smooth muscle cells, η = 11 for collagen) and IL-18BP mice (n = 6 for smooth muscle cells, η = 13 for collagen) were treated. Data represent mean values is s.e.m. Single star means P <0.05; and the two stars indicate P <0.01.

DESCRIPTION OF THE INVENTION

The invention is based on the finding of increased levels of circulating IL-18 in patients, acute coronary syndromes and increased production of IL-18 in unstable carotid atherosclerotic plaques responsible for stroke. In addition, it has been shown that in vivo electrotransfer of an expression DNA plasmid encoding IL-18BP prevents the development of fatty seals in the thoracic aorta and delays the development of advanced atherosclerotic plaques in the aortic sinus in a well-established mouse model. Particularly important, transfection is an IL-18BP plasmid, inducing profound changes in plaque composition (reduction of macrophages, T cells, cell death and lipid content, and increase in smooth muscle cells and collagen content), leading to a stable plaque phenotype . These results indicate for the first time the important role of IL-18 inhibitors in reducing plaque development / progress and promoting plaque stability.

The invention therefore relates to the use of an IL18 inhibitor for the manufacture of a medicament for the treatment and / or prevention of atherosclerosis.

The term "prevention" in the context of this invention refers not only to the complete prevention of any effect, but also to any partial or important prevention, reduction, reduction, reduction or elimination of the effect, before or at the outset of the disease.

The term "treatment" in the context of this invention refers to any beneficial effect in the progression of the disease, including the mitigation, reduction, reduction or elimination of pathological development after the onset of the disease.

The term "IL-18 inhibitor" in the context of this invention refers to any molecule that modulates IL-18 production and / or action in such a way that the production of IL-18 and / or action is attenuated, reduced or substantially reduced is partially or completely prevented or blocked.

The production inhibitor can be any molecule that adversely affects the synthesis, processing or maturation of IL-18. The inhibitors according to the invention may be, for example, suppressors of gene expression of interleukin IL-18, antisense mRNAs that reduce or inhibit the transcription of IL-18 mRNA or lead to mRNA degradation, proteins that impair correct folding, or partially inhibit, or inhibit, the maturation or secretion of IL-18, a protease that breaks down IL-18, once it has already been synthesized and the like. The production inhibitor may be the Caspase-1 inhibitor or the ICE inhibitor, for example by inhibiting the maturation of IL-18.

For example, an inhibitor of the action of IL-18 may be an IL-18 antagonist. The antagonists may either bind to or separate the IL-18 molecule itself with considerable affinity and specificity to neutralize partially or completely the IL-18 or IL-18 binding sites responsible for binding IL-18 to its ligands (e.g., similar to its receptors). The antagonist may also suppress the IL-18 signaling pathway activated in cells after IL-18 receptor binding.

IL-18 inhibitors may also be soluble IL-18 receptors or receptor-like molecules or components that block IL-18 receptors, IL-18 antibodies, for example similar to monoclonal antibodies, or any other component or a molecule that interferes with the binding of IL-18 to its target sites, thereby reducing or preventing target intracellular or extracellular responses mediated by IL-18.

Atherosclerosis is also called arteriosclerosis or hardening of the arteries. In the context of the present invention, the term atherosclerosis includes all diseases or conditions of the arteries, commonly described as atherosclerosis, in which the fatty material is deposited within the walls of the vessels, possibly resulting in narrowing and damage of blood flow, as well as rupture and / or clot formation erosion.

According to the present invention, atherosclerosis involves both sclerosis and loss of elasticity of the arteries due to atheroma (atherosclerosis) or for any other reason (arteriosclerosis). The pathological conditions of atherosclerosis, as well as the complications or consequences of atherosclerosis, which are intended to be included in the term "atherosclerosis" as used herein, are described in detail in the "prior art" above.

The development of atherosclerosis involves the formation of atherosclerotic plaques and their development in more and more unstable forms. Accordingly, the invention also relates to the use of an IL-18 inhibitor for the manufacture of a medicament necessary for reducing or stopping the further development of atherosclerosis.

Vessel obstruction in the formation of atherosclerotic plaque thrombus is a critical event in heart and brain infarctions, which are among the most damaging effects of atherosclerosis. Therefore, the invention also relates to the use of an IL-18 inhibitor for the manufacture of a medicament for the treatment and / or prevention of atherosclerotic plaque thrombosis.

The stability of the plaque affects the development of atherosclerotic plaque toward a harmful or vulnerable plaque, which is a prerequisite for the initiation of thrombosis. Accordingly, the invention further relates to the use of an IL-18 inhibitor for the manufacture of a medicament for the prevention and / or treatment of atherosclerotic plaque instability.

An unstable plaque is prone to rupture, and rupture of the plaque can lead to thrombosis. Accordingly, the invention further relates to the use of an IL-18 inhibitor for the manufacture of a medicament for the prevention of erosion or destruction of atherosclerotic plaque.

Plaque instability and thrombosis may be due, for example, to apoptotic cell death, leading to high procoagulant activity and may be a key event leading to thrombosis of destroyed or ruptured atherosclerotic plaques as well as to embolisms (13, 14). Oxidized lipoproteins (oxLDL) have been shown to induce macrophage and endothelial cell apoptosis in culture (15). As shown in the examples below, it has now been found that an IL-18 inhibitor is capable of greatly reducing the cell death induced by oxLDL.

According to the present invention, it is surprisingly found that blood IL-18 levels are significantly increased in patients with heart failure suffering from recurrent cases, such as death, recurrent ischemia, revascularization, progressive atherosclerosis, or re-hospitalization in cardiac arrest , compared with patients not re-admitted to hospital. This increase in IL18 levels has been specifically reported for those patients who later die, compared to those who survive. Elevated levels of IL-18 in the blood stream are observed in both ischemia patients and non-ischemia patients.

Accordingly, the invention also relates to the use of IL-18 inhibitors for the manufacture of a medicament for the treatment and / or prevention of recurrent heart failure. A recurrent case can be any case after heart failure such as death, recurrent ischemia, re15

/ a * '% your vascularization, progressive atherosclerosis or re-hospitalization for heart failure.

In a preferred embodiment of the invention, heart failure is ischemia, i. due to myocardial ischemia.

In another preferred embodiment, heart failure is not associated with ischemia, such as that due to persistent hypertension, functional heart disease, or pulmonary disease leading to sudden and in this case impaired heart failure.

In a preferred embodiment of the invention, the IL18 inhibitor is selected from the group consisting of ICE inhibitors, antibodies against IL-18, antibodies against any of the subunits of the IL-18 receptor, inhibitors of the IL-18 receptor signaling pathway. IL-18 antagonists that compete with IL-18 and block the receptor for IL-18 and IL-18 binding proteins, isoforms, muteins, recombinant proteins, functional derivatives, active fractions or their cyclic permuted derivatives having the same activity.

As used herein, the term "muteins" refers to analogues of IL-18BP or analogues of viral IL-18BP, in which one or more of the amino acid residues of native IL-18BP or viral IL-18BP are substituted by different amino acid residues, or have eliminated or added one or more amino acid residues to the normal sequence of IL-18BP or viral IL-18BP without significantly altering the activity of the products obtained compared to wild-type IL-18BP or viral IL-18BP. These muteins are prepared by known synthesis methods and / or by site-directed mutagenesis techniques, or any other known techniques suitable therefor.

Preferably, each such mutein has a sequence of amino acids substantially similar to that of IL-18BP or substantially replicates that of viral IL-18BP so that it has substantially similar activity to that of IL-18BP. An activity of IL-18BP is its ability to bind to IL-18. Because mutein has significant activity for binding to IL-18, it can be used to purify IL-18, for example, by means such as affinity chromatography, which means that it can be regarded as having substantially similar IL-18 activity. 18BP activity. Therefore, routine experiments can determine whether a mutein has substantially the same activity as IL-18BP involving the administration of such mutein, for example in competitive sandwich assay, to determine whether it binds or does not bind to a properly labeled IL-18, for example as a radioimmunoassay or ELISA.

The muteins of IL-18BP polypeptides or the muteins of viral IL-18BPs that can be used according to the present invention, or the nucleic acids that encode them, include a substantially defined set of relevant sequences such as replacement peptides or polynucleotides that can be obtained by routine paths from those skilled in the art without having to have a lot of experience, based on the techniques and guidance provided here.

Preferred changes for the muteins of the invention are those known as conservative substitutions. Conservative amino acid substitutions of IL-18BP polypeptides or proteins, or viral IL-18BPs, may include synonymous amino acids in a group that has sufficiently similar physicochemical properties so that substitution between members of the group will retain the biological function of the molecule (16). It is also clear that it is possible to insert and remove amino acids in the sequences defined above without altering their function, especially if insertions or deletions include only a few amino acids, for example below thirty and preferably below ten, such as do not remove or replace amino acids that are critical for functional conformation, such as cysteine residues. Proteins and muteins obtained by such deletions and / or insertions fall within the scope of the present invention.

The synonymous amino acid groups are preferably those defined in Table I. Preferably, the synonymous amino acid groups are those defined in Table II; and even more preferably, the synonymous amino acid groups are those defined in Table III.

TABLE I

Preferred groups of synonymous amino acids

Amino acid

Synonymous group

Sir

Arg

Leu

Pro

Thr

Ala Val

Gly

Sir, Thr, Gly, Asn

Arg, Gin, Lys, Glu, His lie, Phe, Tyr, Met, Val, Leu

Gly, Ala, Thr, Pro

Pro, Ser, Ala, Gly, His, Gin, Thr

Gly, Thr, Pro, Ala

Met, Tyr, Phe, lie, Leu, Val

Ala, Thr, Pro, Ser, Gly

Fri. Met, Tyr, Phe, Val, Leu, lie Phe Trp, Met, Tyr, lie, Val, Leu, Phe Tyr Trp, Met, Phe, He, Val, Leu, Tyr Cys Sir, Thr, Cys His Glu, Lys, Gin, Thr, Arg, His Gin Glu, Lys, Asn, His, Thr, Arg, Gin Asn Gin, Asp, Ser, Asn Lys Glu, Gin, His, Arg, Lys Asp Glu, Asn, Asp Glu Asp, Lys, Asn, Gin, His, Arg, Glu Met Phe, He, Val, Leu, Met

Trp

Trp

TABLE II

More preferred groups of synonymous amino acids

Amino acid

Synonymous group

Sir Sir Arg His, Lys, Arg Leu Leu, He, Phe, Met Pro Yeah, Pro Thr Thr Ala Pro, Ala Val Val, Matt, He Gly Gly He He, Met, Phe, Val, Leu Phe Met, Tyr, lie, Leu, Phe

Tyr Phe, Tyr Cys Cys, Ser His His, Gin, Arg Gin Glu, Gin, His Asn Asp, Asn Lys Lys, Arg Asp Asp, Asn Glu Glu, Gin Met Met, Phe, lie, Vai, Leu Trp Trp

TABLE III

Most preferred groups of synonymous amino acids

Amino acid

Sir

Arg

Leu

Pro

Thr

Ala

Vai

Gly lie

Phe

Tyr

Cys

His

Synonymous group

Sir

Arg

Leu, He, Met

Pro

Thr

Ala

Vai

Gly

He, Matt, Leu

Phe

Tyr

Cys, Ser

His

Gin Gin Asn Asn Lys Lys Asp Asp Glu Glu Met Met, lie, Leu Trp Met

Examples of preparing amino acid substitutions in proteins that can be used to produce muteins of IL-18BP polypeptides or proteins, or muteins of viral IL-18BPs, for use in the present invention include any of the known methodological steps, such as those disclosed in US patents 4,959,314, 4,588,585 and 4,737,462 by Mark et al; No. 5,116,943 to Koths et al., 4,965,195 to Namen et al; No. 4,879,111 to Chong et al; and 5,017,691 from Lee et al; and the lysine-substituted proteins represented in U.S. Pat. No. 4,904,584 (Shaw et al).

The term recombinant protein refers to a polypeptide comprising IL-18BP, or viral IL-18BP, or a mutein associated with another protein that, for example, has existed for a long time in body fluids. Therefore, IL-18BP or viral IL-18BP may be linked to another protein, polypeptide, or the like, such as immunoglobulin or a fragment thereof.

As used herein, the term "Functional Derivatives" encompasses derivatives of IL-18BPs or viral IL-18BP and their muteins and recombinant proteins, which may be obtained from functional groups which occur as side chains in residues or at the N or C terminus groups by methods known in the art and included in the invention as they exist as pharmaceutically acceptable, i. they do not

disrupt the activity of a protein that is substantially similar to IL-18BP activity or viral IL-18BPs and their compositions and have no toxic properties. These derivatives may include, for example, polyethylene glycol side-chains that can mask antigenic sites and prolong the residence of IL-18BP or viral IL-18BP in body fluids. Other derivatives include aliphatic esters of the carboxyl groups, amides of the carboxyl groups by reaction is ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of amino acid residues formed with acyl groups (eg alkanoyl or carboxycyclic aroyl groups) -acyl derivatives of free hydroxyl groups (e.g., those of seryl or threonyl residues) formed with acyl groups.

By the term active fractions of IL-18BP or viral IL-18BP, muteins and recombinant proteins, the present invention encompasses any fragment or precursor of a polypeptide chain of a protein molecule, alone or together with related molecules or residues, e.g. sugar or phosphate residues or aggregates of the protein molecule itself or of the sugar residues themselves, provided that said fraction is substantially similar to IL-18BP activity.

In another preferred embodiment of the invention, the IL-18 inhibitor is an IL-18 antibody. The anti-IL-18 antibodies may be polyclonal or monoclonal, chimeric, human-like, or even fully human. Recombinant antibodies and their fragments are characterized by high binding affinity for IL-18 in vivo and low toxicity. The antibodies that can be used in the invention are characterized by their ability to treat patients for a period sufficient to produce a very good regression or improvement of the pathogenic condition or any symptom or group of symptoms associated with the pathogenic condition and low toxicity.

Neutralizing antibodies can be readily generated in animals, such as rabbits, goats, or mice, by immunization with IL-18. Immunized mice are particularly useful as sources for the delivery of B cells for the industrial production of hybridomas, which in turn are cultured to produce large amounts of anti-IL-18 monoclonal antibodies.

Chimeric antibodies are immunoglobulin molecules that are characterized by two or more segments or moieties derived from different animal species. Typically, the chimeric antibody variable region is derived from a non-human mammalian antibody, such as a murine monoclonal antibody, and the immunoglobulin constant region is derived from a human immunoglobulin molecule. It is preferable that both areas and the combination of these are of low immunogenicity, which can be determined routinely (24). Human-like antibodies are immunoglobulin molecules created by genetically engineered techniques in which murine constant regions are replaced with human copies while maintaining murine antigen-binding regions. The resulting mouse-human chimeric antibody has predominantly reduced immunogenicity and improved human pharmacokinetics. (25).

Therefore, in another preferred embodiment, the IL-18 antibody is a human IL-18 antibody-like antibody. Preferred examples of human-like anti-IL-18 antibodies are described, for example, in European Patent Application EP 0 974 600.

In another preferred embodiment, the IL-18 antibody is fully human. The human antibody production technology is described in detail, for example, in WO00 / 76310, WO99 / 53049, US 6,162,963 or AU 5336100. Fully human antibodies are predominantly recombinant antibodies obtained in transgenic animals, for example xenomice, comprising all or parts of functional human Igku .

In a very preferred embodiment of the present invention, the IL-18 inhibitor is IL-18BP, or isoform, mutein, recombinant protein, functional derivative, active w fraction or a cyclic permuted derivative thereof. These isoforms, muteins, recombinant proteins or functional derivatives, retain the biological activity of IL-18BP, especially binding to IL-18, and preferably have at least activity that is substantially similar to that of IL-18BP. Ideally, these proteins have biological activity that is even higher compared to unmodified IL-18BP. Preferred active fractions have an activity that is better than the activity of IL-18BP, or that have other advantages, such as better stability or lower toxicity or immunogenicity, or they can be obtained more readily in large quantities or yes ** they are easier to clean.

The sequences of IL-18BP and its splice variants / isoforms can be taken from WO99 / 09063 or (12) and (23).

The functional derivatives of IL-18BP can be conjugated to polymers to enhance protein properties such as stability, half-life, bioavailability, human body tolerance or immunogenicity. To achieve this goal, IL18-BP may be coupled to, for example, Polyethylene glycol (PEG). Re-glycolation can be accomplished by the known methods described, for example, in WO 92/13095.

Therefore, in a preferred embodiment of the present invention, IL-18BP is PEOL.

In another preferred embodiment of the invention, the IL-18 inhibitor is a recombinant protein comprising all or part of an IL18 binding protein that is bound to all or part of an immunoglobulin. It is clear to those skilled in the art that the recombinant protein obtained retains the biological activity of IL-18BP, in particular the ability to bind to IL-18. The binding can be done directly or through a short linker peptide, which can be from 1 to 3 amino acid residues in length or longer, for example, 13 amino acid residues in length. Said linker may be a tripeptide, for example with an EFM sequence (GluPhe-Met), or a 13-amino acid linker sequence comprising Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met, inserted between the IL-18BP sequence and the immunoglobulin sequence. The recombinant protein obtained has improved properties, such as prolonged lifespan in body fluids (half-life), increased specific activity, increased expression levels, or facilitated purification of the recombinant protein.

In a preferred embodiment, IL-18BP is bound to the constant region of an Ig molecule. Preferably, it is linked to the heavy chain regions, such as the CH2 and CH3 domains of human IgG1. The production of specific recombinant proteins including IL-18BP and part of an immunoglobulin is described, for example, in Example 11 of WP99 / 09063. Other isoforms of Ig molecules are also suitable for the preparation of recombinant proteins of the present invention, such as IgG ₂ or IgG ₄ isoforms, or other Ig classes, such as

IgM or IgA. The recombinant proteins may be monomeric or multimeric, hetero- or homomultimeric.

Interferons are generally known to have inhibitory effects on viral replication and cell proliferation. For example, interferon-γ plays an important role in aiding the immune and inflammatory responses. Interferon β (IFN-β, type I interferon) is thought to have an anti-inflammatory role.

The invention also relates to the use of a combination of an IL-18 inhibitor and interferon, for the manufacture of a medicament for the treatment of atherosclerosis.

Interferons can also be conjugated with polymers to improve protein stability. Conjugation between interferon β and polyol Polyethylene glycol (PEG) is described, for example, in WO99 / 55377.

In another preferred embodiment of the invention, interferon is Interferon-β (IFN-β), and even more preferably, IFN-β 1a.

The IL-18 inhibitor of production and / or action is preferably used concomitantly, sequentially or separately with interferon.

In another embodiment of the invention, the IL-18 inhibitor is used in combination with a TNF antagonist. TNF antagonists exert their activity in several ways. First, antagonists may bind to or themselves release a TNF molecule with sufficient affinity and specificity to neutralize partially or to a large extent the TNF epitope or epitopes responsible for binding to the TNF receptor (hereinafter referred to as "releasing antagonists" ). For example, a releasing antagonist may be an antibody directed against TNF.

Accordingly, TNF antagonists may suppress the TNF signaling pathway activated by the cell surface receptor after binding is TNF (hereinafter referred to as "signaling antagonists"). Both groups of antagonists are useful either individually or together with the IL-18 inhibitor in the treatment of atherosclerosis.

TNF antagonists are readily identified and evaluated by routine screening of candidates for their effect on native TNF activity on sensitive cell lines in vitro, for example human B cells, in which TNF induces proliferation and immunoglobulin secretion. The assay includes TNF preparation is a different dilution of the candidate antagonist, for example 0.1 to 100 times the molar amount of TNF used in the assay and controls without TNF or is only an antagonist (26).

According to the present invention, releasing antagonists are preferred TNF antagonists for use. Among the releasing antagonists are preferably those polypeptides that bind is high affinity TNF and which have low immunogenicity. Particularly preferred are soluble TNF receptor molecules and neutralizing TNF antibodies. For example, soluble TNF-RI and TNF-RII are useful in the present invention. Shortened forms of these receptors, including extracellular receptor domains or functional portions thereof, are particularly preferred antagonists of the present invention. Shortened soluble TNF type-1 and type-II receptors are described, for example, in EP914431.

Truncated forms of TNF receptors are soluble and have been found in urine and serum such as 30 kDa and 40 kDa TNF inhibitory binding proteins, respectively named TBP I and TBP II (27). According to the invention, the simultaneous, sequential or separate use of the IL-18 inhibitor is preferably a TNF antagonist and / or interferon.

According to the invention, TBP I and TBP II are preferred TNF antagonists for use in combination with an IL-18 inhibitor. In the present invention, derivatives, fragments, portions and biologically active portions of the receptor molecules that functionally resemble receptor molecules may also be used. Such biologically active equivalent or derivative of the receptor molecule refers to a portion of the polypeptide or sequence encoding the receptor molecule that is of sufficient size and is capable of binding TNF with such affinity that the interaction is the membrane-bound TNF receptor is suppressed or blocks.

In another preferred embodiment, the human soluble TNF-RI (TBPI), which is the TNF antagonist of the invention, is used. Natural and recombinantly soluble TNF receptor molecules and methods for their preparation are described in European Patents EP 308 378, EP 398 327 and EP 433 900.

The IL-18 inhibitor may be used concomitantly, sequentially or separately with the TNF inhibitor. The combination of an IL-18 antibody or antiserum and soluble TNF receptor having TNF inhibitory activity is advantageous.

In another preferred embodiment of the invention, the medicament comprises a COX inhibitor, preferably a COX-2 inhibitor. COX inhibitors are known in the art. Specific COX-2 inhibitors are described, for example, in WO 01/00229.

Thromboxane inhibitors, in particular thromboxane A2, are currently widely used for the treatment of atherosclerosis. Therefore, in another preferred embodiment of the invention, the medicament further includes a thromboxane inhibitor, and in particular a thromboxane A2 inhibitor, for simultaneous, sequential or separate use. According to the invention, particularly preferred for use is aspirin in combination with the IL-18 inhibitor.

One of the causes of atherosclerosis seems to be the high concentration of lipids in the blood. Therefore, in another ^w preferred embodiment, the medicament further includes a lipid-reducing component for simultaneous, sequential or separate use. According to the invention, any component known in the art that reduces lipids can be used. Fat-lowering components include drugs such as cholestyramine, colestipol, nicotinic acid, gemfibrozil, probucol, and the like. HMG CoA reductase inhibitors and especially so-called statins are particularly preferred. Many statins, such as Simvastatin or lovastatin, are known in the art.

d 'For the prevention and / or treatment of atherosclerosis, it is even better to use a preferred embodiment of the invention for the use of an IL-18 inhibitor in combination with foods low in fat and / or low in cholesterol and / or low in salt.

In a preferred embodiment of the present invention, the IL-18 inhibitor is used in an amount of from about 0.0001 to 10 mg / kg body weight or from about 0.01 to 5 mg / kg body weight, or from about 0.1 to 3 mg / kg body weight, or from about 1 to 2 mg / kg body weight. In another preferred embodiment, the

Jim.

IL-18 is used in an amount of from about 0.1 to 1000 pg / kg body weight or from 1 to 100 pg / kg body weight, or from about 10 to 50 pg / kg body weight.

The invention further relates to the use of an expression vector comprising the coding sequence for an IL-18 inhibitor in the preparation of a medicament for the prevention and / or treatment of atherosclerosis. Therefore, a gene-therapeutic approach is used to treat and / or prevent the disease. It is advantageous that IL-18 inhibitor expression will be in situ, thereby effectively blocking IL-18 directly in the tissue (s) or cells affected by the disease.

As explained in detail in the examples below, it has been found that effective expression of IL-18BP can be observed in a murine model of the disease after electrotransfer of an expression vector comprising the IL-18BP coding sequence.

Therefore, in a preferred embodiment, the expression vector is administered by electrotransfer, preferably intramuscularly.

According to the invention, it is also contemplated to use a vector for inducing and / or enhancing the endogenous production of an IL-18 inhibitor in a cell that is normally silent for expression of an IL-18 inhibitor or that expresses amounts of the inhibitor that are not sufficient . The vector may include regulatory sequences that function in cells that will express the inhibitor or IL-18. Such regulatory sequences may be, for example, promoters or enhancers. The regulatory sequence may then be introduced into the correct genome locus by homologous recombination, thereby operatively linking the regulatory sequence to the gene whose expression is to be induced or increased. The technology is commonly referred to as "endogenous gene activation" (EGA), and is described, for example, in WO 91/09955.

It is also apparent to those skilled in the art that it is possible to exclude the expression of IL-18 using the same technique, i. by introducing a negative regulatory element, for example, as a silent element at the IL-18 gene locus, which will inhibit regulation or inhibit IL-18 expression. It will be apparent to those skilled in the art that this repressed regulation or silent IL-18 expression will have the same effect as using the IL-18 inhibitor to prevent and / or treat the disease.

The invention further relates to the use of a cell that is genetically modified to produce an IL-18 inhibitor in the manufacture of a medicament for the treatment and / or prevention of atherosclerosis.

The invention further relates to pharmaceutical compositions particularly useful for the prevention and / or treatment of atherosclerosis, which include a therapeutically effective amount of an IL-18 inhibitor and a therapeutically effective amount of interferon. As an inhibitor of IL-18, the composition may include caspase-1 inhibitors, antibodies against IL-18, antibodies against any of the IL-18 receptor subunits, inhibitors of the IL-18 signaling pathway, antagonists of IL-18. that compete with IL-18 and block the receptor of IL-18 and IL-18 binding proteins, isoforms, muteins, recombinant proteins, functional derivatives, active fractions or their cyclic permuted derivatives having the same activity.

IL-18BP and its isoforms, muteins, recombinant proteins, functional derivatives, active fractions or cyclic permuted derivatives, as described above, are the preferred active components of the pharmaceutical compositions.

Preferably, the interferon included in the pharmaceutical composition is IFN-β.

In another preferred embodiment, the pharmaceutical composition comprises therapeutically effective amounts of an IL-18 inhibitor, optionally an interferon and a TNF antagonist. TNF antagonists may be antibodies that neutralize TNF activity, or soluble truncated TNF receptor fragments, also called TBPI and TPVP. The pharmaceutical composition of the invention may further comprise one or more COX inhibitors, preferably COX-2 inhibitors. The pharmaceutical composition of the invention may further include a thromboxane inhibitor such as aspirin and / or a lipid-lowering component, for example as articles.

The term "pharmaceutically acceptable" means that it includes any carrier which does not affect the biological activity of the active ingredient and which is not toxic to the host to which it is administered. For example, for parenteral administration, the active protein (s) may be formulated as a single dosage form for injection in a carrier, such as saline, dextrose solution, serum albumin, and Ringer's solution.

The active ingredients of the pharmaceutical composition according to the invention can be administered to the individual in various ways. Administration routes include intradermal, transdermal (eg, as slow-release agents), intramuscular, intraperitoneal, intravenous, subcutaneous, oral, epidural, topical and intranasal. Any therapeutically effective route of administration, for example absorption through epithelial or endothelial tissue, or through gene therapy, where the patient is administered a DNA molecule encoding the active ingredient (e.g., vector) that results in expression and secretion in vivo of the active component. In addition, the protein (s) of the invention may be administered together with other components of biologically active substances, such as pharmaceutically acceptable surfactants, excipients, carriers, diluents and carriers.

For parenteral administration (e.g., intravenously, subcutaneously, intramuscularly), the active protein (s) may be formulated as a solution, suspension, emulsion or lyophilized powder coupled to a pharmaceutically acceptable parenteral carrier (e.g., water, saline, dextrose solution). and additives that maintain isotonicity (eg mannitol) or chemical stability (eg preservatives and buffers). The preparation is sterilized by standard techniques.

The bioavailability of the active protein (s) according to the invention can also be improved by using conjugation methods that increase the half-life of the molecule in the human body, such as binding of the molecule to polyethylene glycol, as described in PCT Patent Application WO 92 / 13095.

The therapeutically effective amounts of the active protein (s) will be a function of many variables including the type of antagonist, the affinity of the antagonist for IL-18, any residual cytotoxic activity that the antagonist has, the route of administration, the clinical condition of the patient (including desire for the patient) maintenance of non-toxic level of endogenous IL-18 activity).

A therapeutically effective amount is that amount which, when administered, the IL-18 inhibitor results in inhibition of the biological activity of IL-18. The individual dose administered as a single or multiple dose will vary depending on various factors including the pharmacokinetic properties of the IL18 inhibitor, routes of administration, patient status and characteristics (gender, age, body weight, health, judgment), grade symptoms, concurrent treatments, frequency of treatment and desired effect. Adjustment and manipulation of established dose ranges are well known to those skilled in the art as well as in vitro and in vivo methods for determining the inhibition of IL-18 in an individual.

According to the invention, the IL-18 inhibitor is used in an amount of about 0.0001 to 10 mg / kg or about 0.01 to 5 mg / kg body weight, or about 0.01 to 5 mg / kg body weight, or about 0.1 to 3 mg / kg body weight, or about 1 to 2 mg / kg body weight. Other preferred amounts of IL-18 inhibitors are amounts of about 0.1 to 1000 gg / kg body weight, or about 1 to 100 gg / kg body weight, or about 10 to 50 gg / kg body weight.

The method of administration which is preferred according to the invention is subcutaneous administration. Another preferred route of the invention is intramuscular administration.

In other preferred embodiments, the IL-18 inhibitor is administered daily or every other day.

Daily doses are usually given as two separate doses or in the form of sustained-release effective doses to obtain the desired results. Second or subsequent applications may be made at the same dose less than or greater than the original or previous dose administered to the individual. A second or subsequent application may be made during or before the onset of the disease.

According to the invention, the IL-18 inhibitor may be administered to the patient prophylactically or therapeutically, before, concurrently or sequentially with other therapeutic regimens or preparations (e.g., multi-drug regimens), in a therapeutically effective amount. The active ingredients, which are administered concomitantly with other therapeutic agents, may be administered with the same or different compositions.

The invention further relates to a method of treating and / or preventing atherosclerosis, comprising administering to a host in need thereof an effective inhibitory amount of an IL-18 inhibitor.

The invention further relates to a method of preventing and / or treating atherosclerosis comprising administering to a host in need thereof an expression vector comprising the coding sequence of the IL-18 inhibitor.

In a preferred embodiment of the invention, the expression vector is administered continuously, and more preferably by intramuscular injection.

The invention further relates to the use of IL-18 as a diagnostic marker for poor clinical prognosis in heart failure. Poor clinical prognosis involves any worsening of the patient's condition, such as recurrent conditions, or even death, followed by myocardial infarction.

It is preferable to use IL-18 as a diagnostic marker for recurrent conditions after the first case of heart failure. Periodic conditions include, but are not limited to, recurrent ischemia, re-vascularization, progressive atherosclerosis, or re-hospitalization for heart failure.

Having described the invention, it will be much better understood by the following examples which enable it to be illustrated and in no way intended to limit the present invention.

EXAMPLES FOR THE IMPLEMENTATION OF THE INVENTION

Materials and methods

Samples

Forty-one human atherosclerotic plaques were obtained from 36 patients undergoing carotid endarterectomy. As controls, 2 carotid and 3 internal mammary arteries were obtained, without atherosclerosis (2 with minimal fibromuscular seals), at autopsy, or during surgical coronary bypass surgery. They were immediately placed in liquid nitrogen and stored at -80 ° C. The plaques used for protein and RNA extraction were rapidly rinsed and placed in liquid nitrogen before being stored at -80 ° C. For immunohistochemical studies, the plates are placed in fresh 4% paraformaldehyde for 2 hours, then transferred into 30% sucrose-PBS solution before freezing and breaking at the optimum cutting temperature in the required medium (OSC Compound , Miles Inc, Diagnostics Division) with liquid nitrogen and stored at -80 ° C for cryostat sections. Several sections of 8- to 10-μm thickness were obtained from each sample for histological analysis and immunohistochemical studies.

Classification of patients

To investigate the possible relationship between IL-18 / IL-18βP expression and signs of plaque instability, we collected, in a suspected and random manner, clinical data from 23 consecutive patients (out of 36) undergoing endarterectomy between May and August 2000. Information on the presence or absence of intra-plaque ulcers in macroscopic examination is submitted on a continuous basis by the surgeon who conducts the endarterectomy procedure. This allows the plates to be classified as ulcers with or without ulcers. In addition, patients were classified according to clinical symptoms into two separate groups. Patients who have clinical symptoms of cerebral ischemic attack related to carotid stenosis are classified as symptomatic. Endarterectomy is performed 2-66 days (17.6 ± 5.3 days) after the onset of clinical symptoms in these patients. Patients who have never had symptoms of cerebral ischemia in the carotid artery are classified as asymptomatic. Asymptomatic carotid stenoses are detected on the basis of C systemic clinical studies in patients with coronary or peripheral disease, the severity of the disease being determined using periodic Doppler ultrasound by an experienced, certified ultrasound examiner. Although asymptomatic patients have never had a case of ischemia in the area of carotid stenosis, carotid endarterectomy has been shown to be favorable for these patients, according to researchers (28) in the Asymptomatic Carotid Atherosclerotic Study (ACAS).

Western blot analysis

Proteins from 12 atherosclerotic plaques and 5 control normal arteries were extracted. The frozen samples were destroyed under liquid nitrogen. The powder samples were resuspended in ice-cold lysis buffer [20 mmol / L Tris-HCl, pH 7.5, 5 mmol / L EGTA, 150 mmol / L NaCl, 20 mmol / L glycerophosphate, 10 mmol / L NaF, 1 mmol / L sodium orthovanadate, 1% Triton X-100, 0.1% Tween 20, 1 pg / mL aprotinin, 1 mmol / L PMSF, 0.5 mmol / L N-TO3mi-Lζ * phenylalanine chloromethyl ketone (TPCK), 0.5 mmol / LN (a ) -p-TO3myl-lysine chloromethyl ketone (TLCK)] at a ratio of 0.3 mL / 10 mg wet weight. The extracts were incubated on ice for 15 minutes and then centrifuged (12,000 g, 15 minutes, 4 ° C). Detergent soluble supernatant fractions were maintained and protein concentrations in the samples were equilibrated using Bio-Rad protein assay.

For Western blot analysis for IL-18 and IL-18R, protein extracts were boiled for 5 minutes and applied to 7.5% or 15% SDS-polyacrylamide gel. IL-18BP, rhIL-18 purified from E.Coli (Serono Pharmaceutical Research W Institute, Geneva), was bound to Affligel 15 (Biorad) 1 mg / ml of resin according to the manufacturer's protocol. Protein extracts (60 pg) were incubated overnight at 4 ° C on a roller with 20 μΐ of resin adjusted to 500 μΐ with PBS 0.05% Tween. To remove any nonspecific binding, the resin was centrifuged and washed with 10 mM Tris pH 8, 140 mM NaCl, 0.5% Triton-X-100 (Fluka), 0.5% deoxycholate, then 50 mM Tris pH 8,200. mM NaCl, 0.05% TX100, 0.05% .Nonidate P40 (Fluka), 2 mM CHAPS (Boehringer, Mannheim), followed by a final wash with 50 mM Tris, pH 8. The resin was then centrifuged, resuspended in the sample buffer. under reducing conditions, it was boiled for 5 minutes and finally applied to a 10% SDS polyacrylamide NuPAGE gel (Invitrogen).

Samples were electrophoretically transferred from the polyacrylamide gel to nitrocellulose. The nitrocellulose membranes were saturated for 2 hours at room temperature in TBST [50 mmol / L Tris-HCl (pH 7.5), 250 mmol / L NaCl, and 0.1% Tween saline] containing 5% skimmed milk powder. The membranes were then incubated with goat anti-human IL-18 and IL-18R (α-chain) polyclonal antibodies (1 pg / ml) (R&D Systems), a murine anti-human IL-18 BP monoclonal antibody (MAb 657.27 with 5 pg / ml) (Corbaz et al., 2000 article presented), rabbit anti-human caspase-1 polyclonal antibody (1 pg / ml) (A-19, Santa Cruz). The specificity of MaB 657.27 was analyzed on a cut membrane strip using a competitor using 200 x molar excess of rhIL-18BP-6his (purified from Chinese hamster ovary cells, Serono Pharmaceutical Research Institute), coincubated with Mab 657.27 with 5 Lg / ml. , for 1 hour. After incubation with the appropriate HRP conjugated antibodies, chemiluminescent substrates (ECL, Western blotting; Amersham Corp) were used to show positive bands according to the manufacturer's instructions, and the bands were visualized after exposure to Hyperfilm ECL (Amersham Corp).

Immunohistochemistry

Frozen sections of 6 atherosclerotic plaques were incubated with 1:10 normal horse serum or 1:10 normal goat serum for 30 minutes at room temperature, washed once with PBS, then incubated or with the first mouse monoclonal antibody against CD68 for macrophage identification (DAKO-CD68, KR1), or with the first murine monoclonal antibody against human smooth muscle α-actin (1A4, DAKO), to identify smooth muscle cells. Specific goat polyclonal antibodies (R&D Systems) were used to identify the IL-18 and IL-18 receptors in atherosclerotic plaques at a dilution of 5 pg / mL. IL-18 BP was detected using a specific monoclonal antibody directed against recombinant human IL-18BP isoform (H2O) (Corbaz et al., 2000 article presented). After washing with PBS, the sections were incubated with the following second biotinylated antibodies: biotinylated equine anti-mouse IgG (Vector Laboratories, Inc), at a dilution of 1: 200, to detect staining with antibodies against CD68, smooth muscle α-actin and IL- 18 BP, and biotinylated equine anti-goat IgG (Vector), at a dilution of 1: 200, for detection of anti-IL-18 and anti-IL-18 receptor antibodies. Immunoblotting was visualized using avidinbiotin HRP imaging systems (Vectastain ABC kit PK-6100 Vector). Adjacent sections were stained as negative controls with w-isotype-like unbound antibodies instead of primary antibodies.

RNA preparation

From 29 atherosclerotic plaques, total RNA was extracted into acid guanidine thiocyanate solution and extracted with phenol and chloroform according to the method of Chomczynski and Sacchi (29). The purified RNA was dissolved in water and the concentration and measured by absorbance at 260 nm. RNA integrity was assessed by electrophoresis on a 1% agarose gel. The cDNA was synthesized from pg total PHK using the Promega reverse transcriptase system according to the manufacturer's protocol.

Semi-quantitative and Real-time PCR of human IL-18 and IL18BP in human atherosclerotic plaques

Semi-quantitative PCR reactions were performed in a total volume of 50 μΐ, in the presence of 1U of AmpliTaq DNA Polymerase (Perkin Elmer, Roche, U.S.A), 2.5 mM dNTPs (Amersham, U.S.A) and 50 pmoles of forward and reverse PCR primers. The reactions were incubated in PTC200 Peltier Effect Thermal Cycler (MJ Research, U.S.A) under the following conditions: denaturation for 1 minute at 94 ° C, hybridization for 1 minute at 5 5 ° C, and continued reaction for 1 minute at 72 ° C. To ensure comparison of the amount of PCR products during the linear phase of the PCR reaction, IL-18BP, IL-18 and β-actin were analyzed after 25, 28 and 31 cycles. The optimum number of cycles for IL-18BP, IL-18 and β-actin was determined before saturation of the bands (31, 28 and 25, respectively). PCR primers were constructed based on published sequences (AF110799, D49950, X00351) as follows:

IL-18,

IL18BP, β-actin, reverse straight line reverse reverse line off

5′-GCGTCACTACACTCAGCTAA-3 ′; 5'-GCCTAGAGGTATGGCTGTAA-3 ';

'-ACCTGTCTACCTGGAGTGAA-3';

'-GCACGAAGATAGGAAGTCTG-3';

'-GGAGGAGCAATGATCTTGATCTTC-3';

'-GCTC ACC ATGGATGATGATATCGC-3'. amplification of possible genomic DNA,

To contaminate the samples, PCR reactions were performed in the absence of a cDNA template. PCR products (10µ1) were analyzed on 1% agarose gels by electrophoresis in 1X TAE buffer. The size of

PCR products were checked by comparison with a 1 kb ladder (Gibco) after staining of the calves. The relative amount of ethidium bromide strips under UV light is evaluated using Kodak Digital Sciences analytical software and reported as the ratio of the target gene (hIL-18BP, hIL-18) to the housekeeping gene (Ιιβ-actm) .

SYBR Green Real Time PCR primers were designed for IL18, IL-18BP and GAPDH (house-keeping control) using Primer Express software from PE Biosystems according to published sequences (AF110799, D49950, NM 002046) as follows: IL-18 , reverse 5'-CAGCCGCTTTAGCAGCCA-3 ';

rights 5'-CAAGGAATTGTCTCCCAGTGC-3 ';

IL 18BR reverse 5 '-ACACAGGCTTGAGCGTTCC-3'; rights 5 '-TCCCATGTCTCTGCTCATTTAGTC-3';

GAPDH, reverse 5′-GATGGGATTTCCATTGATGACA-3 ′;

rights 5'-CCACCCATGGCAAATTCC-3 ';

intron-GAPDH reverse 5′-CCTAGTCCCAGGGCTTTGATT-3 ′; rights 5 '-CTGTGCTCCC ACTCCTGATTTC-3'.

Specificity and optimal primer concentration are analyzed. Possible genomic DNA contamination is ruled out by conducting the PCR reaction with specific intronGAPDH primers. The absence of non-specific amplification was confirmed by electrophoretic analysis of PCR products with 3.5% agarose gel. SYBR Green Real-Time PCR was performed with 5 μΐ / well of RT products (0.5 ng total RNA), 25 μΐ / well of SYBR Green PCR master mix (PE Biosystem, CA, USA), with AmpErase Uracil N-Glycosylase ( UNG) (0.5 Unit / well) and 20 μΐ primers (300 nM). PCR was performed at 50 ° C for 2 minutes (for AmpErase UNG incubation to remove any uracil included in the cDNA),

95 ° C for 10 min (for AmpliTaq Gold activation) and then performing 40 cycles at 95 ° C for 15 seconds, 60 ° C for 1 minute with the ABI PRISM 7700 Detection System. In this way, cDNA samples are amplified by reverse transcriptase and their Ct (threshold cycle) values determined. All Ct values were normalized to the housekeeping gene GAPDH. Single specific DNA strand for IL-18, IL-18BP and GAPDH was observed using gel electrophoretic analysis.

The principle of real-time detection using the "SYBR Green PCR master mix" is based on the direct detection of the PCR product by measuring the increased fluorescence caused by the binding of SYBR Green dye to double stranded DNA.

Statistical analysis

Data are given as mean ± SEM. IL-18 levels were compared between groups using the Mann-Whitney test. A value of p 0.05 was considered statistically significant.

Example 1: Oxidative Lipoprotein Induced Inhibitors of IL-18 Inhibitors of Endothelial Cell Death (oxLDL)

Cultured human umbilical vein endothelial cells (HUVECs) were exposed for 16 hours to oxLDL, in the presence or absence of an IL-18 binding protein or anti-1E-18 antibody. As shown in Figure 1, 83% of HUVECs die after exposure to oxLDL. Co-incubation with IL18BP or with anti-IL-18 antibody saves the cells almost completely from death. No cell death was observed using IL18BP. 89% of cells survive when an anti-IL-18 antibody is used.

This experiment clearly demonstrates the protective effect of two different IL-18 inhibitors against cell death due to apoptosis in atherosclerotic plaque.

Example 2: Expression of IL-18 Protein and its Endogenous IL-18 BP Inhibitor in Atherosclerotic Plates

Western blot analyzes of protein extracts from 12 carotid atherosclerotic arteries and 5 normal controls were performed. The active-form protein IL-18 is strongly expressed in all atherosclerotic plaques until almost no expression is detected or it is completely missing in normal arteries (Figure 2). Starts 1 to 4 contain samples of atherosclerotic plaques, starts 5 to 7 are from normal arteries. Interestingly, the detection of the active form of IL-18 probably correlates with the expression of the active form of caspase-1, which is involved in the processing of IL-18 (Figure 2, fourth row). Significant expression of the IL-18 receptor protein (overexpression) was also observed in all atherosclerotic plaques, compared to the very low expression level in normal arteries (Figure 2, second row). In addition, most of the atherosclerotic plaques express IL-18BP, although the expression level is heterogeneous (Figure 2 first row).

Example 3: Cellular localization of the IL-18 protein and its endogenous IL-18BP inhibitor in atherosclerotic plaques

Immunohistochemical studies were performed with 6 carotid atherosclerotic plaques to determine the cellular localization of IL-18 and IL-18BP. As can be seen from Figure 2, IL-18 is mainly expressed in macrophages, these cells being probably the major source of IL-18 in the plaque (not shown). These areas are also rich in POP positive lymphocytes. However, T lymphocytes do not appear to be directly involved in the production of IL-18. IL-18 is also expressed in some internal smooth muscle cells and less frequently in endothelial cells. In contrast, significant expression of IL-18BP was detected in capillary endothelial cells with plaques and in those of the lumen surface, although expression was not detected in all vessels. Relatively lower and more heterogeneous expression of IL-18 BP is also detected, mainly extracellularly, in some macrophage-rich regions.

Example 4: Expression of IL-18 and IL-18BP mRNA transcripts in atherosclerotic plaques and association with plaque instability

To determine whether human IL-18 and IL-18BP mRNA were expressed in human carotid atherosclerotic plaques, semi-quantitative RT-PCR was performed with six atherosclerotic plaques (Figure 3). IL-18 and IL-18BP mRNAs are detected in all atherosclerotic plaques, although the amount of mRNA for IL18 and IL-18BP is heterogeneous. Therefore, to accurately calculate the levels of IL-18 and IL-18BP mRNA expression, 23 atherosclerotic plaques were further analyzed using the SYBR Green Real-Time PCR method (Figure 4). The plaques are characterized by clinical and pathological examination as symptomatic (unstable) or asymptomatic (stable) plaques containing macroscopically or not containing ulcers. The clinical characteristics of the patients are summarized in Table 4. The percentage reduction in carotid diameter (60% - 95%) and risk factors including age, diabetes, hypercholesterolemia, hypertension and smoking did not differ between the two groups.

Table 4

Characteristics of the patient

Asymptomatic Symptomatic Patients (n = 9) Patients ¹ (n = 14) Age 66.9 ± 4.0 70.2 ± 3.9 Genus Male (8) Men's (9) Hypertension 8 9 Hypercholesterolemia ³ 4 8 Diabetes 3 1 Currently smoking 7 8 Coronary artery disease 5 4 * These patients are with temporary or persistent ischemic

cerebral attack 2-66 days before endarterectomy.

² Number of patients with clinical hypertension treated with antihypertensive components.

³ Number of patients with clinical hypercholesterolemia who have been treated with lipid-lowering drugs.

The amount of IL-18 was found to be up-regulated in symptomatic versus asymptomatic atherosclerotic plaques (2.03 ± 0.5 vs. 0.67 ± 0.17, respectively) (Figure 4A). Statistical analysis showed that this increase in IL-18 production observed in symptomatic plaques was of high significance (p <0.0074), whereas the increased amount of IL-18BP observed in symptomatic versus asymptomatic plaques was not (4.64 ± 0.98 vs. 2.5 ± 0.92, respectively) (Figure 4B). In other words, although both symptomatic and asymptomatic groups show a positive correlation between IL-18 and IL-18BP mRNA, the slopes are significantly different between the two groups (symptomatic group: slope 1.16 [0.19-2.14], r ² = 0.36 versus asymptomatic group: slope 4.79 [2.39-7.20], r ² = 0.76; p <0.05). Therefore, it seems that the relative increase in IL-18BP expression in the symptomatic group is not sufficient to compensate for the increased expression of IL-18. Furthermore, since the presence of inflammation is considered as a distinguishing feature of plaque instability, a statistical analysis of plaques with or without intra-plaque inflammation was further performed and a marked upregulation of IL-18 in plaques was demonstrated. ulcers (p <0.018) were present (Figure 4C).

These data indicate that the increase in IL18 expression observed in atherosclerotic plaques correlates with plaque instability.

Example 5: IL-18BP modulates atherosclerotic lesion development and stability in in vivo disease models

Methods

Characteristics of the patient

Plasma samples are obtained from patients with acute ischemic coronary syndromes (unstable angina and myocardial infarction) less than 7 days after the onset of symptoms. Unstable angina is defined as associated with typical chest pain, or with ischemic changes in the electrocardiogram, or with the presence of coronary artery disease. Myocardial infarction is diagnosed on the basis of typical ischemic changes in the electrocardiogram associated with a significant increase in myocardial enzymes (creatine phosphokinase and troponin I) in the circulating blood. Patients without ischemia are recruited to the same cardiology department and are completely deprived of ischemia signals. Plasma levels of human IL-18 were determined using a commercially available kit (MBL, Japan).

In vivo intramuscular electrotransfer of mouse IL-18BP expression plasmid

Fourteen male C57BL / 6 apoE KO mice, 14 weeks of age, received, at intervals of three weeks, 3 injections of the expression IL-18BP plasmid, pcDNA3-IL18BP. Control mice (n = 19) were injected with a control blank plasmid. The mouse IL-18BP isoform d cDNA isolated as described (accession number # Q9ZOM9) (23) was subcloned into EcoR1 / Not1 sites from an expression vector of a mammalian pcDNA3 cell, under the control of a cytomegalovirus promoter (Invitrogen). The construct termed 334.yh is shown in Figure 5. Similarly, a control plasmid devoid of therapeutic cDNA was constructed. IL-18BP or the control expression plasmid (60 Rg) was injected into both the tibial and cranial muscles of the anesthetized mouse as previously described (13). Briefly, electrical impulses through the skin (8 rectangular (square) electrical impulses 200 V / cm, 20 msec duration, 2 Hz) are supplied by a PS-15 electropulsor (Genetronics, France) using two stainless steel flat electrodes mounted 4.2 to 5.3 mm from each other on each side of the foot.

Elisa mIL-18BP

The plates were coated overnight with the r-mlL-18BPO-affinity purified rabbit polyclonal antibody (5 pg / well). Soluble mIL-18BP was detected using a biotinylated rabbit polyclonal antibody (0.3 pg / ml) prepared against E. coli r-mIL-18BP (Peprotec), followed by extravidine peroxidase (1/1000) (Sigma). The bound rabbit polyclonal antibody was analyzed by Western blot to confirm the specificity of mIL-18BP. Recombinant mIL-18BPd derived from HEK 293 cells was used as a standard. ELISA sensitivity was 5 ng / ml.

Mouse analysis

Cryostatic sections (8 pm) were obtained from the aortic sinus and used for the detection of lipid deposition using Oil red, for collagen detection using Sirius red, and for immunohistochemical analysis as described previously (13). The sections were stained with specific first antibodies: anti-mouse macrophage, clone MOMA2 (BioSource), conjugated alkaline phosphatase anti-α-actin for smooth muscle cells and anti-CD3 for

T lymphocytes (Dako) as previously described (13). The use of the TUNEL technique (13) has been used to detect cell death. CD3 positive cells were counted microscopically by chance. Atherosclerotic plaques in the aortic sinus and areas stained positive for macrophages, smooth muscle cells, collagen or TUNEL were measured using a computer generated quantitative image assay (NS15000, Microvision) as previously described (13). Staining with non-immune isotype-like immunoglobulins determines the specificity of the immunostaining. The specificity of TUNEL is determined by the release of the terminal deoxynucleotidyl transferase enzyme. Thoracic aortas covering the left subclavian artery to the renal arteries were fixed with 10% buffered formalin and stained for lipid deposition with Oil red. They are then opened lengthwise and the percentage of lipid deposition calculated using a computer generated image quantification image (NS15000, Microvision).

Results

X; This study analyzes the hypothesis that regulation of IL-18 / IL-18BP plays a critical role for both atherosclerosis and plaque stability. Plasma IL-18 levels were measured in patients with acute coronary syndromes (30 men, 18 women, mean age 66.2 ± 1.8 years, of whom 14 had unstable angina and 34 had myocardial infarction) and in control patients without ischemia, recruited from the same cardiology department (10 males, 3 females, mean age 60.0 ± 5.2 years). Plasma IL-18 levels increased significantly in patients with acute coronary disease compared to controls (146.9 ± 17.1 vs. 73.0 ± 12.2 pg / ml, respectively, p <0.05) compared to circulating IL-18BP levels, which were slightly increased (20.1 ± 2.7 versus 7.5 ± 2.5 ng / ml, respectively, p = 0.06). In addition, IL-18 levels correlate with the severity of the disease, with the highest levels observed in patients with severe ischemic heart dysfunction and clinical reports of pulmonary edema (224.03 ± 39.1 pg / ml, p <0.001 compared to controls) . These results, obtained from patients with acute heart disease, together with previous observations that IL-18 increases in atherosclerotic plaques in patients with stroke [Mallat, 2001], suggest a possible important role for the regulation of IL-18 / IL-18BP in atherosclerotic process.

Therefore, we tested this hypothesis using apoE knockout (KO) mice that develop spontaneously similar to human, atherosclerotic lesions. Fourteen mice, 14 weeks of age, received in addition IL-18BP intramuscularly, by in vivo electrotransfer of plasmid DNA encoding murine IL-18BPd, while 19 controls of similar age received an empty plasmid. Plasmid electrotransfer was repeated every 3 weeks and mice were killed 23 weeks at 9 weeks post-treatment. Mouse IL-18BP plasma levels were lower than the limit (5 ng / ml) in apoE KO mice injected with the empty plasmid. However, a single injection of IL-18BP plasmid resulted in high levels of IL-18BP in the blood, with a maximum increase of 2 days after injection (323.5 ± 100.9 ng / ml) and 127.4 ± 35.4 ng / ml measured after 2 weeks. Nine weeks after treatment, either IL-18BP or blank plasmid, total cholesterol (489.4 ± 34.6 vs. 480.8 ± 36.3 mg / dl, respectively) and serum levels of high-density lipoproteins (52.3 ± 9.4 vs. 48.8 ± 5.1 mg / dl , respectively) are no different between the two groups. A moderate but significant increase in animal weight was observed in the IL-18BP treated group compared to the control group (31.8 ± 0.9 versus 28.6 ± 0.8 g, respectively, p <0.05).

The result of the addition of IL-18BP in atherosclerosis was evaluated in two different sections: the descending thoracic aorta and the aortic sinus. The thoracic aorta was selected to determine the role of IL-18BP in the development of fatty compaction (atherogenesis), since thoracic atherosclerotic lesions were absent almost completely at 14 weeks of age (data not shown) where IL-18BP transfection began. The aortic sinus, where atherosclerotic lesions are already present at 14 weeks of age (data not shown), is examined for advanced plaque progression and composition, an important factor for plaque stability. IL-18BP treatment of apoE KO mice significantly affects the development and progression of the atherosclerotic lesion. Thoracic aorta examination showed a significant reduction in lipid deposition in mice treated with the IL-18BP plasmid compared to the empty plasmid (Figure 6). A computer-generated quantitative analysis image showed a 69% reduction in the degree of atherosclerotic lesions (p <0.0001) (Figure 6), indicating the critical, decisive role of IL-18 in atherosclerosis. In addition, treatment with the IL-18BP plasmid for only 9 weeks significantly reduced the development of advanced atherosclerotic plaques in the aortic sinus (24% reduction in plaque size, p = 0.01) compared to the treatment with the empty plasmid (Figure 7).

Much more importantly, the composition of advanced lesions, as a major factor in plaque instability, is highly influenced by treatment with IL-18BP. Atherosclerotic lesions of mice treated with the IL-18BP plasmid showed a significant 50% reduction in macrophage infiltration (p <0.0001) (Figure 8) containing 67% less T lymphocytes (p <0.005) (Figure 8) increased accumulation in smooth muscle cells (p <0.05) (Figure 9). In addition, these significant changes in the damaged cellular composition were associated with a significant 85% increase in collagen content (p <0.0005), which was determined by staining with Sirius red and a decrease in total lipid content.

Therefore, treatment with IL-18BP leads to a significant reduction in the inflammatory process in atherosclerotic lesions and induces a healing process, a characteristic of stable atherosclerotic plaques. In addition, the marked reduction in the inflammatory component of lesions in IL18BP-treated mice is associated with a significant reduction in plaque cell death (2.9 ± 0.9% in IL-18BP treated mice relative to

10.5 ± 3.6% in controls, p <0.05), thereby reducing the enlargement and thrombogenicity of the acellular lipid core [Mallat, 1999].

Conclusions:

Using a well established murine model resembling human atherosclerosis, the results reported above clearly demonstrate the unexpected and critical role of IL-18 and IL-18BP regulation in the development, progression and stability of atherosclerotic plaques. Like the prevention of early lesion formation in the thoracic aorta, inhibition of IL-18 activity by the addition of IL-18BP also strongly affects the composition of advanced lesions in the aortic sinus, inducing a switch to a stable plaque phenotype.

Clinical prognosis in patients with atherosclerosis depends only partially on the size of the lesions. The idea that quality (the composition of the plaque) rather than the size of the lesion may be an even better prophet for the incidence of ischemic cases is increasingly accepted today. Indeed, the severe clinical manifestations of atherosclerosis (heart and brain infarctions) are mainly due to obstruction of the vascular lumen by a thrombus that forms at the site of contact of the destroyed atherosclerotic plaque (19). Pathologic studies have shown that vulnerable or unstable plaques that are prone to destruction or destroyed are rich in inflammatory cells and show significant loss of smooth muscle cells and contained in collagen (20, 21). In addition, such plaques show a significant increase in apoptotic cell death, leading to the formation of a highly thrombogenic lipid core (13, 22). It is noteworthy that all of these plaque instability signals decrease significantly in IL-18BP-treated mice, indicating that IL-18 signaling is a major factor in plaque instability.

The practical relevance of the results obtained in apoE KO mice with respect to human disease is enhanced by our findings for increased levels of circulating IL-18 in patients with acute coronary syndromes and increased production of IL-18 in unstable carotid atherosclerotic plaques, responsible for stroke. Taken together, these findings identify IL-18 activity inhibitors as novel important therapeutics for preventing and treating the development of atherosclerotic plaques and limiting complications in their development.

Example 6: Elevated IL-18 levels correlate with recurrent cases of patients with heart failure

IL-18 levels are measured in the blood serum of patients by ELSIA with an IL-18 specific antibody.

All 56 ischemically ill patients, as well as patients without ischemia, heart failure or patients who do not have one, are analyzed.

In patients who died later, IL-18 levels were 216.0 + 41.5 pg / ml, compared to 112.2 + 12.2 pg / ml in non-lethal patients (p = 0.0018).

In patients who experience any recurrent cases, such as death, recurrent ischemia, revascularization, progressive atherosclerosis, or re-hospitalization for heart failure, the following IL-18 levels were measured: 165.8 + 23.8 versus 107.7 + 14.6, at patients who did not experience recurrent cases (p = 0.03).

These results indicate that IL-18 levels are significantly elevated in patients with poor clinical prognosis, such as those with recurrent conditions or even in those who die.

IL-18 levels were measured in the blood samples of 16 patients who did not suffer from ischemic disease, who had or who lacked heart failure. In patients who die late, the levels are 199.0 + 34.8 pg / ml versus 95.3 + 20.4 pg / ml, in those patients who remain alive (p = 0.09).

Patients with any recurrent conditions: 146.6 + 34.4 pg / ml IL-18 versus 95.4 + 23.9 pg / ml in patients who did not experience recurrent cases (p = 0.03). Although differences in IL-18 levels are not statistically significant, due to the small number of patients, there is a clear tendency for an increase in IL-18 levels.

In patients with ischemia, IL-18 levels were 214.2 + 45.9 pg / ml in patients who died, compared to 118.4 + 12.8 pg / ml in those who remained alive (p = 0.007).

In patients with any recurrent condition, the measured IL-18 levels were 162.8 + 24.7 pg / ml compared to 116.2 + 16.0 in patients who did not experience recurrent conditions.

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USING IL-18 INHIBITORS FOR THE TREATMENT AND / OR PREVENTION OF ATHEROSCLEROSIS (Abstract)

The invention relates to the use of an IL-18 inhibitor for the manufacture of a medicament for the treatment and / or prevention of atherosclerosis.

2235/02-GP

USE OF IL-18 INHIBITORS FOR THE TREATMENT AND / OR PREVENTION OF ATHEROSCLEROSIS

TECHNICAL FIELD

The present invention is in the field of vascular diseases. In particular, the invention relates to the use of IL-18 inhibitors for the treatment and / or prevention of atherosclerosis.

BACKGROUND OF THE INVENTION

Atherosclerosis is a widespread and very important vascular disease, but many other vascular disorders are known. Basically, atherosclerosis affects the large arteries and those of medium size and lesions and includes fatty seals, phobrolytic plaques and lesions with complications.

Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by the progressive accumulation of lipids such as cholesterol, cells such as macrophages, T lymphocytes or smooth muscle cells and the extracellular matrix (1). Large deposits are called atheromas or plaques that often contain calcium. The adipose tissue can destroy the wall of the artery, reducing the elasticity of the artery and thus obstructing blood flow. Possibly, blood clots may form around the deposited plaques, which further impede blood flow, which can lead to a general blockage of the blood vessel. Typically, atherosclerosis is associated with increased levels of LDL-cholesterol, Lp (a) fibrinogen and factor VII, as well as decreased levels of HDL-cholesterol. Risk factors include increased age, male, smoking, diabetes, obesity, high blood cholesterol, foods rich in fat and humans, individually or family-laden with heart disease. It most commonly causes organ ischemia, such as myocardial infarction.

Atheroma is a widespread damage to the arteries, which may be further complicated by thrombo-embolism. Atheromatous plaques often constrict the lumen of the arteries, causing ischemia, and sometimes atrophy of tissues in the hypoperfusion region. Serious consequences include the symptom of angina due to myocardial ischemia, heart failure due to ischemia or non-ischemic cases, and hypertension due to renal artery narrowing and renal hypoperfusion, whose physiological response is increased secretion .

Sometimes atherosclerosis and arteriosclerosis are referred to as separate pathological conditions and in this case, atherosclerosis is defined as involving hardening (sclerosis) or loss of elasticity of the arteries due to atheroma, whereas arteriosclerosis is hardening or loss of hardness for any other reason.

Complications or sequelae of atherosclerosis include coronary artery disease (coronary artery atherosclerosis), insufficient blood supply due to obstruction (ischemia / angina,) acute MI (myocardial infarction, heart attack), transient ischemic attack, or stroke blood vessels, muscles or body organs.

Aneurysms, which are permanent, abnormal dilations of blood vessels, are also common consequences of atherosclerosis. Atherosclerotic abdominal aortic aneurysms usually develop in adult patients. They may burst into the retroperitoneal space. In atherosclerotic aneurysms there is usually a pronounced loss of elastic tissue and fibrosis of the media, mainly due to ischemia of the aortic muscle muscle, followed by the release of macrophage enzymes leading to fragmentation of the elastic fibers.

Drugs that are recommended for the treatment or prevention of atherosclerosis include lowering fat / cholesterol in the blood. In particular, the therapy for lowering LDL-cholesterol is widely used. Statins, which are specific inhibitors of HMG CoA reductase, are currently most widely used. In addition, other fat-lowering components include drugs such as cholestyramine, colestipol, nicotinic acid, gemfibrozil, probucol, lovastatin, and the like.

Another approach is to reduce the risk of blood clots in atheromatous lesions. Aspirin, which is considered to be a specific inhibitor of thromboxane A2 mediated platelet aggregation or anticoagulant, may be used to reduce the risk of thrombosis.

Subcutaneous balloon angioplasty uses a balloon-tip catheter to smooth out plaques and increase blood flow through obstructed areas. The technique is similar to that used to open the arteries of the heart, but it can also be applied to many other arteries in the body. Coronary artery stenosis is bypassed with portions of a soap vein, stitched with the proximal aorta, or by dissection of the internal mammary artery from the chest wall and joining the distal end with an artery from the anterior surface of the heart.

Surgical debridement (endarterectomy) may only be recommended in some cases (for example: endarterectomy of the carotid artery).

However, the main recommendation is to treat or control risk factors such as maintaining low fat, low cholesterol and low salt foods and to follow health recommendations for the treatment and control of hypertension, diabetes and other diseases, body weight and smoking cessation, as well as periodic examination to determine the normal state and functioning of the heart.

The inflammatory process is involved in all the different stages of atherosclerosis (1). The first controllable stage of atherosclerosis is thought to be endothelial activation by various factors, including weaker cell contact, modified lipoproteins, and pro-inflammatory cytokines (1). Many studies have recently shown that interactions between vascular cells and those at the site of inflammation are critical for atherosclerosis (1). In particular, suppression of certain pre-inflammatory pathways reduces the development of atherosclerosis (1).

Inflammation plays a major role in the destruction of atherosclerotic plaques and thrombosis (2 - 5) and therefore influences cases of acute ischemic syndromes and associated mortality (6). In fact, the severe clinical manifestations of atherosclerosis, including heart, brain, and other organs affected by atherosclerosis, are mainly due to obstruction of the vascular lumen by thrombi resulting from contact with the destroyed atherosclerotic plaque (3, 4).

Pathological studies have shown that vulnerable or unstable plaques, i. plaques prone to tearing or already tearing differ greatly in cellular composition and matrix composition compared to stable non-tear-resistant plaques (7). Vulnerable plaques are abundant in inflammatory cells (macrophages and T lymphocytes) and contain a thrombogenic lipid core and are characterized by a fine fibrotic coating with significant loss of extracellular matrix (7).

Reduced collagen synthesis mediated by the proinflammatory cytokine IFNγ and increased activity of macrophage-derived matrix degrading metalloproteinases are responsible for the thinning and brittleness of the fibrotic coating (7). The rupture of the fragile fibrous coating strongly exposes the thrombogenic lipid core in the circulating blood and leads to the formation of thrombosis (1, 7). Therefore, cell density due to inflammation in an atherosclerotic lesion is considered to be a good indication of its instability.

The clinical prognosis for a patient with atherosclerosis depends only partially on the size of the lesion (19; 20). Today it is widely accepted that quality (composition of the plaque) rather than the size of the lesion may even be a better indicator of the occurrence of ischemic cases. In fact, severe clinical manifestations of atherosclerosis (heart and brain infarctions) are mainly due to obstruction of the vascular lumen due to thrombus formation at the contact of the destroyed atherosclerotic plaque (19). Pathological studies have shown that vulnerable or unstable plaques that are prone to tearing or torn are rich in inflammatory cells and show significant loss of smooth muscle cells and collagen content (20, 21). Furthermore, such plaques show a significant increase in apoptotic cell death leading to the formation of a highly thrombogenic lipid core (13, 22).

Προ-inflammatory cytokines are involved in inflammation.

The cytokine interleukin 18 (IL-18) was originally described as a γ interferon (IFN-γ) inducing factor (8). It is an early signal in the development of T-lymphocyte helper cell type 1 (Th1) responses. IL-18 works in conjunction with IL-12, IL-2, antigens, mitogens, and probably other factors to induce IFN-γ production. IL-18 also increases the production of GM-CSF and IL-2, potentiates anti-CD3 induced T cell proliferation and enhances Fas-mediated destruction of natural killer cells. Mature IL-18 is derived from its precursor by the IL1β conversion enzyme (ICE, caspase-1). The IL-18 receptor consists of at least two components that cooperate in ligand binding. High- and low-affinity binding sites for IL-18 are C found in murine IL-12 stimulated T cells (9), suggesting a multi-stranded receptor complex. So far, two receptor subunits have been identified, both belonging to the IL-1 receptor family (10).

IL-18 signal transduction involves activation of NF-κB (11).

Recently, soluble protein has been isolated from human urine, which has a strong affinity for IL-18, and human and mouse cDNAs have been cloned as well as the human gene (12; WO 99/09063). The protein is referred to as the IL-18 binding protein (IL-18BP).

IL18BP is not an extracellular domain of one of the known IL18 receptors but a secreted, naturally circulating protein. It belongs to a new family of secreted proteins, further comprising several Poxvirus-encoded proteins (12). IL18BP is continuously expressed in the spleen (12). The urine protein, as well as the recombinant IL18BP, bind specifically to high affinity IL-18 and modulate the biological affinity of IL-18.

The IL18BP gene is located in human chromosome 1 lql3 and no exon encoding a transmembrane domain is found in the 8.3 kb genomic sequence. Four splice variants or isoforms of IL18BP have been identified in humans and are designated IL18BP a, b, c and d, all having the same N-terminus and differing at their C-ends (12).

Four human and two mouse IL-18BP isoforms derived from mRNA splicing and found in various cDNA libraries have been expressed, purified and evaluated for binding and neutralizing the biological activities of IL-18 (23). The human IL-18BP isoform a (IL-18BPa) exhibits the highest affinity for IL-18 with a fast on and off rate and a dissociation constant (K (d)) of 399 pM. IL-18BP shares the Ig domain of IL-18BP a, with the exception of the C-terminal 29 amino acids; K (d) of IL-18BP c was 10-fold lower (2.94 nM). However, IL-18BP a and IL-18BP neutralize IL-18> 95% with a molar excess of both. The isoforms of IL-18BP b and IL-18BP d do not have a complete Ig domain and are not capable of binding or neutralizing IL-18. Mouse IL-18BP c and IL-18BP d isoforms have the identical Ig domain and also neutralize> 95% murine IL-18 with a molar excess of both. However, murine IL-18BP d, which has a common C-terminal motif with human IL-18BP?, Also neutralizes human IL-18. Molecular modeling identifies large mixed electrostatic and hydrophobic binding sites in the Ig domain of IL18BP, which may explain its strong affinity for ligand binding (23).

SUMMARY OF THE INVENTION

The invention is based on the finding that the IL-18 inhibitor has a distinct beneficial effect on plaque development, plaque progression and their stability in a murine model of atherosclerosis. The IL-18 inhibitor not only prevents lesion formation in the thoracic aorta, but also induces a switch to a stable plaque phenotype in already established atherosclerotic plaques.

The invention therefore relates to the use of an IL-18 inhibitor for the manufacture of a medicament for the prevention and / or treatment of atherosclerosis. In addition, the invention relates to methods of treatment, by gene therapy approach, for the treatment and / or prevention of atherosclerosis.

BRIEF DESCRIPTION OF THE GRAPHS

Figure 1 is a histogram showing the survival rate of human umbilical vein endothelial cells after incubation with oxidized lipoproteins alone, or incubated with a combination of oxidized lipoproteins and IL-18 antibody or IL 18BP respectively.

Figure 2 shows a Western blot performed with protein extracts from atherosclerotic arteries compared to control arteries. Antibodies against IL-18BP (hIL18BP), the IL-18 receptor α subunit (hIL18Ra), IL-18 (hIL18) and Caspase-1 (Caspase-1 p10) were used in Western blotting.

Figure 3 shows an ethidium bromide agarose gel showing the result of RT-PCR analysis for IL-18 and IL-18BP mRNA in atherosclerotic plaque cells.

Figure 4 Typical RT-PCR results for IL-18BP and IL-18 in atherosclerotic plaques compared with ha-actin (control) expression in symptomatic and asymptomatic plaques.

Figure 5 shows a map of the expression vector used for intramuscular electrotransfer in mice.

Figure 6 is a histogram showing the stained lipid region in atherosclerotic arteries. Computer generated image for quantitative analysis of lipid deposition. Data represent mean values with s.e.m. (η = 19 for empty plasmid, η = 14 for IL-18BP plasmid). The four stars indicate P <0.0001.

Figure 7 is a histogram showing a lesion of the aortic sinus region after IL-18BP treatment compared to the control (empty plasmid). Computer generated image for quantitative analysis of the damaged area. Data represent mean values with s.e.m. (η = 19 for empty plasmid, η = 14 for IL-18BP plasmid). The two stars indicate P <0.01.

Figure 8 shows the effect of IL-18BP treatment on the content of damaged inflammatory cells. A computer-generated image for quantitative analysis was used to determine the percentage of macrophage-positive areas (black stripes) and the number of infiltrated T lymphocytes per mm (gray lines) in aortic sinus lesions from the control (n = 12 for macrophage staining, η = 15 for T lymphocyte staining) or IL-18BP treated mice (n = 13 for macrophages, η = 12 for T lymphocytes). Data represent mean values with s.e.m. The three stars indicate P <0.005; and the four stars indicate P <0.0001.

Figure 9 shows the effect of IL-18BP treatment on smooth muscle cell lesion and collagen content. A computer-generated image for quantitative analysis was used to determine the percentage of the positive region of smooth muscle cell (black lines) and collagen accumulation (gray lines) in aortic sinus lesions from control (n = 6 for smooth muscle cells, η = 11 for collagen) and treated with IL-18BP mice (n = 6 for smooth muscle cells, η = 13 for collagen). Data represent mean values with s.e.m. Single star means P <0.05; and the two stars indicate P <0.01.

DESCRIPTION OF THE INVENTION

The invention is based on the finding of increased levels of circulating IL-18 in patients with acute coronary syndromes and increased production of IL-18 in unstable carotid atherosclerotic plaques responsible for stroke. In addition, it has been shown that in vivo electrotransfer of an expression DNA plasmid encoding IL-18BP prevents the development of fatty seals in the thoracic aorta and delays the development of advanced atherosclerotic plaques in the aortic sinus in a well-established mouse model. It is particularly important that transfection with the IL-18BP plasmid induces profound changes in plaque composition (reduction of macrophages, T cells, cell death and lipid content and increase in smooth muscle cells and collagen content), leading to a stable plaque phenotype . These results indicate for the first time the important role of IL-18 inhibitors in reducing plaque development / progress and promoting plaque stability.

The invention therefore relates to the use of an IL18 inhibitor for the manufacture of a medicament for the treatment and / or prevention of atherosclerosis.

The term "protection" in the context of this invention refers not only to the complete prevention of any effect, but also to any partial or important prevention, reduction, reduction, reduction or elimination of the effect, before or at the outset of the disease.

The term "treatment" in the context of this invention refers to any beneficial effect in the progression of the disease, including the mitigation, reduction, reduction or elimination of pathological development after the onset of the disease.

The term "IL-18 inhibitor" in the context of this invention refers to any molecule that modulates IL-18 production and / or action in such a way that the production of IL-18 and / or action is attenuated, reduced or substantially reduced is partially or completely prevented or blocked.

The production inhibitor can be any molecule that adversely affects the synthesis, processing or maturation of IL-18. The inhibitors according to the invention may be, for example, suppressors of gene expression of interleukin IL-18, antisense mRNAs that reduce or inhibit the transcription of IL-18 mRNA or lead to mRNA degradation, proteins that impair correct folding, or partially inhibit, or inhibit, the maturation or secretion of IL-18, a protease that breaks down IL-18, once it has already been synthesized and the like. The production inhibitor may be the Caspase-1 inhibitor or the ICE inhibitor, for example by inhibiting the maturation of IL-18.

For example, an inhibitor of the action of IL-18 may be an IL-18 antagonist. The antagonists may either bind to or separate the IL-18 molecule itself with considerable affinity and specificity to neutralize partially or completely the IL-18 or IL-18 binding sites responsible for binding IL-18 to its ligands (e.g., similar to its receptors). The antagonist may also suppress the IL-18 signaling pathway activated in cells after IL-18 receptor binding.

IL-18 inhibitors may also be soluble IL-18 receptors or receptor-like molecules or components that block IL-18 receptors, IL-18 antibodies, for example similar to monoclonal antibodies, or any other component or a molecule that interferes with the binding of IL-18 to its target sites, thereby reducing or preventing target intracellular or extracellular responses mediated by IL-18.

Atherosclerosis is also called arteriosclerosis or hardening of the arteries. In the context of the present invention, the term atherosclerosis includes all diseases or conditions of the arteries, commonly described as atherosclerosis, in which the fatty material is deposited within the walls of the vessels, possibly resulting in narrowing and damage of blood flow, as well as rupture and / or erosion is the formation of clots.

According to the present invention, atherosclerosis involves both sclerosis and loss of elasticity of the arteries due to atheroma (atherosclerosis) or for any other reason (arteriosclerosis). The pathological conditions of atherosclerosis, as well as the complications or consequences of atherosclerosis, which are intended to be included in the term "atherosclerosis" as used herein, are described in detail in the "prior art" above.

The development of atherosclerosis involves the formation of atherosclerotic plaques and their development in more and more unstable forms. Accordingly, the invention also relates to the use of an IL-18 inhibitor for the manufacture of a medicament necessary for reducing or stopping the further development of atherosclerosis.

Vessel obstruction in the formation of atherosclerotic plaque thrombus is a critical event in heart and brain infarctions, which are among the most damaging effects of atherosclerosis. Therefore, the invention also relates to the use of an IL-18 inhibitor for the manufacture of a medicament for the treatment and / or prevention of atherosclerotic plaque thrombosis.

The stability of the plaque affects the development of atherosclerotic plaque toward a harmful or vulnerable plaque, which is a prerequisite for the initiation of thrombosis. Accordingly, the invention further relates to the use of an IL-18 inhibitor for the manufacture of a medicament for the prevention and / or treatment of atherosclerotic plaque instability.

An unstable plaque is prone to rupture, and rupture of the plaque can lead to thrombosis. Accordingly, the invention further relates to the use of an IL-18 inhibitor for the manufacture of a medicament for the prevention of erosion or destruction of atherosclerotic plaque.

Plaque instability and thrombosis may be due, for example, to apoptotic cell death, leading to high procoagulant activity and may be a key event leading to thrombosis of destroyed or ruptured atherosclerotic plaques as well as to embolisms (13, 14). Oxidized lipoproteins (oxLDL) have been shown to induce macrophage and endothelial cell apoptosis in culture (15). As shown in the examples below, it has now been found that an IL-18 inhibitor is capable of ** largely reducing the cell death induced by oxLDL.

According to the present invention, it is surprisingly found that blood IL-18 levels are significantly increased in patients with heart failure suffering from recurrent cases, such as death, recurrent ischemia, revascularization, progressive atherosclerosis, or re-hospitalization in cardiac arrest , compared with patients not re-admitted to hospital. This increase in IL18 levels has been specifically reported for those patients who later die compared with those who survive. Elevated levels of IL-18 in the blood stream are observed in both ischemia patients and non-ischemia patients.

Accordingly, the invention also relates to the use of IL-18 inhibitors for the manufacture of a medicament for the treatment and / or prevention of recurrent heart failure. A recurrent case can be any case after heart failure such as death, recurrent ischemia, re15

vascularization, progressive atherosclerosis, or re-hospitalization for heart failure.

In a preferred embodiment of the invention, heart failure is ischemia, i. due to myocardial ischemia.

In another preferred embodiment, heart failure is not associated with ischemia, such as that due to persistent hypertension, functional heart disease, or pulmonary disease leading to sudden and in this case impaired heart failure.

In a preferred embodiment of the invention, the IL18 inhibitor is selected from the group consisting of ICE inhibitors, antibodies against IL-18, antibodies against any of the subunits of the IL-18 receptor, inhibitors of the IL-18 receptor signaling pathway. IL-18 antagonists that compete with IL-18 and block the receptor for IL-18 and IL-18 binding proteins, isoforms, muteins, recombinant proteins, functional derivatives, active fractions or their cyclic permuted derivatives having the same activity.

As used herein, the term "muteins" refers to analogues of IL-18BP or analogues of viral IL-18BP, in which one or more of the amino acid residues of native IL-18BP or viral IL-18BP are substituted by different amino acid residues, or have eliminated or added one or more amino acid residues to the normal sequence of IL-18BP or viral IL-18BP without significantly altering the activity of the products obtained compared to wild-type IL-18BP or viral IL-18BP. These muteins are prepared by known synthesis methods and / or by site-directed mutagenesis techniques, or any other known techniques suitable therefor.

Preferably, each such mutein has a sequence of amino acids substantially similar to that of IL-18BP or substantially replicates that of viral IL-18BP in such a way that it has substantially similar activity to that of IL-18BP. An activity of IL-18BP is its ability to bind to IL-18. Because mutein has significant activity for binding to IL-18, it can be used to purify IL-18, for example, by means such as affinity chromatography, which means that it can be regarded as having substantially similar IL-18 activity. 18BP activity. Therefore, routine experiments can determine whether a mutein has substantially the same activity as IL-18BP involving the administration of such mutein, for example in competitive sandwich assay, to determine whether it binds or does not bind to a properly labeled IL-18, for example as a radioimmunoassay or ELISA.

The muteins of IL-18BP polypeptides or the muteins of viral IL-18BPs that can be used according to the present invention, or the nucleic acids that encode them, include a substantially defined set of relevant sequences such as replacement peptides or polynucleotides that can be obtained by routine paths from those skilled in the art without having to have a lot of experience, based on the techniques and guidance provided here.

Preferred changes for the muteins of the invention are those known as conservative substitutions. Conservative amino acid substitutions of IL-18BP polypeptides or proteins, or viral IL-18BPs, may include synonymous amino acids in a group that has sufficiently similar physicochemical properties so that substitution between members of the group will retain the biological function of the molecule (16). It is also clear that it is possible to insert and remove amino acids in the sequences defined above without altering their function, especially if insertions or deletions include only a few amino acids, for example below thirty and preferably below ten, such as do not remove or replace amino acids that are critical for functional conformation, such as cysteine residues. Proteins and muteins obtained by such deletions and / or insertions fall within the scope of the present invention.

The synonymous amino acid groups are preferably those defined in Table I. Preferably, the synonymous amino acid groups are those defined in Table I; and even more preferably, the synonymous amino acid groups are those defined in Table III.

TABLE I

Preferred groups of synonymous amino acids

Amino acid Synonymous group

Sir Sir, Thr, Gly, Asn Arg Arg, Gin, Lys, Glu, His Leu He, Phe, Tyr, Met, Val, Leu Pro Gly, Ala, Thr, Pro Thr Pro, Ser, Ala, Gly, His, Gin, Thr Ala Gly, Thr, Pro, Ala Val Met, Tyr, Phe, lie, Leu, Val Gly Ala, Thr, Pro, Ser, Gly

Fri. Phe Tyr Cys His Gin Asn Lys Asp Glu Met Trp Met, Tyr, Phe, Vai, Leu, lie Trp, Met, Tyr, lie, Vai, Leu, Phe Trp, Met, Phe, He, Vai, Leu, Tyr Ser, Thr, Cys Glu, Lys, Gin, Thr, Arg, His Glu, Lys, Asn, His, Thr, Arg, Gin Gin, Asp, Ser, Asn Glu, Gin, His, Arg, Lys Glu, Asn, Asp Asp, Lys, Asn, Gin, His, Arg, Glu Phe, He, Vai, Leu, Met Trp TABLE II

More preferred groups of synonymous amino acids

Amino acid Synonymous group Sir Arg Leu Pro Thr Ala Vai Gly lie Phe Sir His, Lys, Arg Leu, He, Phe, Met Yeah, Pro Thr Pro, Ala Wai, Matt, He Gly lie, Met, Phe, Vai, Leu Met, Tyr, He, Leu, Phe

Tyr Phe, Tyr Cys Cys, Ser His His, Gin, Arg Gin Glu, Gin, His Asn Asp, Asn Lys Lys, Arg Asp Asp, Asn Glu Glu, Gin Met Met, Phe, lie, Val, Leu Trp Trp

TABLE III

Most preferred groups of synonymous amino acids

Amino acid

Sir

Arg

Leu

Pro

Thr

Ala

Val

Gly

He

Phe

Tyr

Cys

His

Synonymous group

Sir

Arg

Leu, lie, Met

Pro

Thr

Ala

Val

Gly

He, Matt, Leu

Phe

Tyr

Cys, Ser

His

Gin Gin Asn Asn Lys Lys Asp Asp Glu Glu Met Matt, He, Leu Trp Met

Examples of preparing amino acid substitutions in proteins that can be used to produce muteins of IL-18BP polypeptides or proteins, or muteins of viral IL-18BPs, for use in the present invention include any of the known methodological steps, such as those disclosed in US patents 4,959,314, 4,588,585 and 4,737,462 by Mark et al; No. 5,116,943 to Koths et al., 4,965,195 to Namen et al; No. 4,879,111 to Chong et al; and 5,017,691 from Lee et al; and the lysine-substituted proteins represented in U.S. Pat. No. 4,904,584 (Shaw et al).

The term recombinant protein refers to a polypeptide comprising IL-18BP, or viral IL-18BP, or a mutein associated with another protein that, for example, has existed for a long time in body fluids. Therefore, IL-18BP or viral IL-18BP may be linked to another protein, polypeptide, or the like, such as immunoglobulin or a fragment thereof.

As used herein, the term Functional Derivatives encompasses derivatives of IL-18BPs or viral IL-18BP and their muteins and recombinant proteins, which may be obtained from functional groups which occur as side chains in residues or in N- or C-terminal groups , by methods known in the art and included in the invention, since they exist as pharmaceutically acceptable, i.e. they do not impair the activity of a protein that is actually similar to activity of IL-18BP, or of viral IL-18BPs and their compositions and does not have toxic properties. These derivatives may include, for example, polyethylene glycol side-chains that can mask antigenic sites and prolong the residence of IL-18BP or viral IL-18BP in body fluids. Other derivatives include aliphatic esters of the carboxyl groups, amides of the carboxyl groups by reactions with ammonia or primary or secondary amines, N-acyl derivatives of free amino groups of amino acid residues formed with acyl groups (e.g. alkanoyl or carboxycyclic aroyl groups) -acyl derivatives of free hydroxyl groups (e.g., those of seryl or threonyl residues) formed with acyl groups.

By the term active fractions of IL-18BP or viral IL-18BP, muteins and recombinant proteins, the present invention encompasses any fragment or precursor of a polypeptide chain of a protein molecule, alone or together with related molecules or residues, e.g. sugar or phosphate residues or aggregates of the protein molecule itself or of the sugar residues themselves, provided that said fraction is substantially similar to IL-18BP activity.

In another preferred embodiment of the invention, the IL-18 inhibitor is an IL-18 antibody. Ahth-IL-18 antibodies can be polyclonal or monoclonal, chimeric, human-like, or even fully human. Recombinant antibodies and their fragments are characterized by high binding affinity for IL-18 in vivo and low toxicity. The antibodies that can be used in the invention are characterized by their ability to treat patients for a period sufficient to produce a very good regression or improvement of the pathogenic condition or any symptom or group of symptoms associated with the pathogenic condition and low toxicity.

Neutralizing antibodies can be readily generated in animals, such as rabbits, goats, or mice, by immunization with IL-18. Immunized mice are particularly useful as sources for the delivery of B cells for the industrial production of hybridomas, which in turn are cultured to produce large amounts of anti-IL-18 monoclonal antibodies.

Chimeric antibodies are immunoglobulin molecules that are characterized by two or more segments or moieties derived from different animal species. Typically, the chimeric antibody variable region is derived from a non-human mammalian antibody, such as a murine monoclonal antibody, and the immunoglobulin constant region is derived from a human immunoglobulin molecule. It is preferable that both areas and the combination of these are of low immunogenicity, which can be determined routinely (24). Human-like antibodies are immunoglobulin molecules created by genetically engineered techniques in which murine constant regions are replaced with human copies while maintaining murine antigen-binding regions. The resulting mouse-human chimeric antibody has predominantly reduced immunogenicity and improved human pharmacokinetics. (25).

Therefore, in another preferred embodiment, the IL-18 antibody is a human IL-18 antibody-like antibody. Preferred examples of human-like anti-IL-18 antibodies are described, for example, in European Patent Application EP 0 974 600.

In another preferred embodiment, the IL-18 antibody is fully human. The human antibody production technology is described in detail, for example, in WO00 / 76310, WO99 / 53049, US 6,162,963 or AU 5336100. Fully human antibodies are predominantly recombinant antibodies obtained in transgenic animals, for example xenomice, comprising all or parts of functional human Igku .

In a very preferred embodiment of the present invention, the IL-18 inhibitor is IL-18BP, or isoform, mutein, recombinant protein, functional derivative, active fraction, or cyclic permuted derivative thereof. These isoforms, muteins, recombinant proteins or functional derivatives, retain the biological activity of IL-18BP, especially binding to IL-18, and preferably have at least activity that is substantially similar to that of IL-18BP. Ideally, these proteins have biological activity that is even higher compared to unmodified IL-18BP. Preferred active fractions have an activity that is better than the activity of IL-18BP, or that have other advantages, such as better stability or lower toxicity or immunogenicity, or they can be obtained more readily in large quantities or to be easier to purify.

The sequences of IL-18BP and its splice variants / isoforms can be taken from WO99 / 09063 or (12) and (23).

The functional derivatives of IL-18BP can be conjugated to polymers to enhance protein properties such as stability, half-life, bioavailability, human body tolerance or immunogenicity. To achieve this goal, IL18-BP may be coupled to, for example, Polyethylene glycol (PEG). Resyclication can be accomplished by known methods described, for example, in WO 92/13095.

Therefore, in a preferred embodiment of the present invention, IL-18BP is PEOL.

In another preferred embodiment of the invention, the IL-18 inhibitor is a recombinant protein comprising all or part of an IL18 binding protein that is bound to all or part of an immunoglobulin. It is clear to those skilled in the art that the recombinant protein obtained retains the biological activity of IL-18BP, in particular the ability to bind to IL-18. The binding may be made directly or by a short linker peptide, which may be from 1 to 3 amino acid residues in length or longer, for example, 13 amino acid residues in length. Said linker may be a tripeptide, for example with an EFM sequence (GluPhe-Met), or a 13-amino acid linker sequence comprising Glu-Phe-Gly-Ala-Gly-Leu-V al-Leu-Gly-Gly-Gln-Phe-Met inserted between the IL-18BP sequence and the immunoglobulin sequence. The recombinant protein obtained has improved properties, such as prolonged lifespan in body fluids (half-life), increased specific activity, increased expression levels, or facilitated purification of the recombinant protein.

In a preferred embodiment, IL-18BP is a bound constant region of an Ig molecule. Preferably, it is linked to the heavy chain regions, such as the CH2 and CH3 domains of human IgG1. The production of specific recombinant proteins including IL-18BP and part of an immunoglobulin is described, for example, in Example 11 of WP99 / 09063. Other isoforms of Ig molecules are also suitable for the preparation of recombinant proteins of the present invention, such as IgG? or IgG ₄ , or other Ig classes, such as

IgM or IgA. The recombinant proteins may be monomeric or multimeric, hetero- or homomultimeric.

Interferons are generally known to have inhibitory effects on viral replication and cell proliferation. For example, interferon-γ plays an important role in aiding the immune and inflammatory responses. Interferon β (IFN-β, type I interferon) is thought to have an anti-inflammatory role.

The invention also relates to the use of a combination of an IL-18 inhibitor and interferon, for the manufacture of a medicament for the treatment of atherosclerosis.

Interferons can also be conjugated with polymers to improve protein stability. Conjugation between interferon β and polyol Polyethylene glycol (PEG) is described, for example, in WO99 / 55377.

In another preferred embodiment of the invention, interferon is Interferon-β (IFN-β), and even more preferably, IFN-β 1a.

The IL-18 inhibitor of production and / or action is preferably used concomitantly, sequentially or separately with interferon.

In another embodiment of the invention, the IL-18 inhibitor is used in combination with a TNF antagonist. TNF antagonists exert their activity in several ways. First, antagonists may bind to or themselves release a TNF molecule with sufficient affinity and specificity to neutralize partially or to a large extent the TNF epitope or epitopes responsible for binding to the TNF receptor (hereinafter referred to as "releasing antagonists" ). For example, a releasing antagonist may be an antibody directed against TNF.

Accordingly, TNF antagonists may suppress the TNF signaling pathway activated by the cell surface receptor after binding to TNF (hereinafter referred to as "signaling antagonists"). Both groups of antagonists are useful either individually or together with the IL-18 inhibitor in the treatment of atherosclerosis.

TNF antagonists are readily identified and evaluated by routine screening of candidates for their effect on native TNF activity on sensitive cell lines in vitro, for example human B cells, in which TNF induces proliferation and immunoglobulin secretion. The assay includes a TNF preparation with different dilutions of the candidate antagonist, for example 0.1 to 100 times the molar amount of TNF used in the assay and controls without TNF or with the antagonist alone (26).

According to the present invention, releasing antagonists are preferred TNF antagonists for use. Among the releasing antagonists are preferably those polypeptides that bind to TNF with high affinity and which have low immunogenicity. Particularly preferred are soluble TNF receptor molecules and neutralizing TNF antibodies. For example, soluble TNF-RJ and TNF-RII are useful in the present invention. Shortened forms of these receptors, including extracellular receptor domains or functional portions thereof, are particularly preferred antagonists of the present invention. Shortened soluble TNF type-1 and type-II receptors are described, for example, in EP914431.

Truncated forms of TNF receptors are soluble and have been found in urine and serum such as 30 kDa and 40 kDa TNF inhibitory binding proteins, respectively named TBP I and TBP II (27). According to the invention, the simultaneous, sequential or separate use of the IL-18 inhibitor with the TNF antagonist and / or interferon is preferably.

According to the invention, TBP I and TBP II are preferred TNF antagonists for use in combination with an IL-18 inhibitor. In the present invention, derivatives, fragments, portions and biologically active portions of the receptor molecules that functionally resemble receptor molecules may also be used. Such a biologically active equivalent or derivative of the receptor molecule refers to a portion of the polypeptide or sequence encoding the receptor molecule that is of sufficient size and capable of binding to TNF with such affinity that the interaction with the membrane-bound TNF receptor is inhibited or blocks.

In another preferred embodiment, the human soluble TNF-RI (TBPI), which is the TNF antagonist of the invention, is used. Natural and recombinantly soluble TNF receptor molecules and methods for their preparation are described in European Patents EP 308 378, EP 398 327 and EP 433 900.

The IL-18 inhibitor may be used concomitantly, sequentially or separately with the TNF inhibitor. The combination of an IL-18 antibody or antiserum and soluble TNF receptor having TNF inhibitory activity is advantageous.

In another preferred embodiment of the invention, the medicament comprises a COX inhibitor, preferably a COX-2 inhibitor. COX inhibitors are known in the art. Specific COX-2 inhibitors are described, for example, in WO 01/00229.

Thromboxane inhibitors, in particular thromboxane A2, are currently widely used for the treatment of atherosclerosis. Therefore, in another preferred embodiment of the invention, the medicament further includes a thromboxane inhibitor, and in particular a thromboxane A2 inhibitor, for simultaneous, sequential or separate use. According to the invention, particularly preferred for use is aspirin in combination with the IL-18 inhibitor.

One of the causes of atherosclerosis seems to be the high concentration of lipids in the blood. Therefore, in another preferred embodiment, the medicament further includes a lipid-lowering component for simultaneous, sequential or separate use. According to the invention, any component known in the art that reduces lipids can be used. Fat-lowering components include drugs such as cholestyramine, colestipol, nicotinic acid, gemfibrozil, probucol, and the like. Particularly preferred are HMG Co A reductase inhibitors, and especially so-called statins. Many statins, such as Simvastatin or lovastatin, are known in the art.

For the prevention and / or treatment of atherosclerosis, it is even better to use a preferred embodiment of the invention for the use of an IL-18 inhibitor in combination with foods low in fat and / or low in cholesterol and / or low in salt.

In a preferred embodiment of the present invention, the IL-18 inhibitor is used in an amount of from about 0.0001 to 10 mg / kg body weight or from about 0.01 to 5 mg / kg body weight, or from about 0.1 to 3 mg / kg body weight, or from about 1 to 2 mg / kg body weight. In another preferred embodiment, the

IL-18 is used in an amount of from about 0.1 to 1000 pg / kg body weight or from 1 to 100 pg / kg body weight, or from about 10 to 50 pg / kg body weight.

The invention further relates to the use of an expression vector comprising the coding sequence for an IL-18 inhibitor in the preparation of a medicament for the prevention and / or treatment of atherosclerosis. Therefore, a gene-therapeutic approach is used to treat and / or prevent the disease. It is advantageous that IL-18 inhibitor expression will be in situ, thereby effectively blocking IL-18 directly in the tissue (s) or cells affected by the disease.

As explained in detail in the examples below, it has been found that effective expression of IL-18BP can be observed in a murine model of the disease after electrotransfer of an expression vector comprising the IL-18BP coding sequence.

Therefore, in a preferred embodiment, the expression vector is administered by electrotransfer, preferably intramuscularly.

According to the invention, it is also contemplated to use a vector for inducing and / or enhancing the endogenous production of an IL-18 inhibitor in a cell that is normally silent for expression of an IL-18 inhibitor or that expresses amounts of the inhibitor that are not sufficient . The vector may include regulatory sequences that function in cells that will express the inhibitor or IL-18. Such regulatory sequences may be, for example, promoters or enhancers. The regulatory sequence may then be introduced into the correct genome locus by homologous recombination, thereby operatively linking the regulatory sequence to the gene whose expression is to be induced or increased. The technology is commonly referred to as "endogenous gene activation" (EGA), and is described, for example, in WO 91/09955.

It is also apparent to those skilled in the art that it is possible to exclude the expression of IL-18 using the same technique, i. by introducing a negative regulatory element, for example, as a silent element at the IL-18 gene locus, which will inhibit regulation or inhibit IL-18 expression. It will be apparent to those skilled in the art that this repressed regulation or silent IL-18 expression will have the same effect as using the IL-18 inhibitor to prevent and / or treat the disease.

The invention further relates to the use of a cell that is genetically modified to produce an IL-18 inhibitor in the manufacture of a medicament for the treatment and / or prevention of atherosclerosis.

The invention further relates to pharmaceutical compositions particularly useful for the prevention and / or treatment of atherosclerosis, which include a therapeutically effective amount of an IL-18 inhibitor and a therapeutically effective amount of interferon. As an inhibitor of IL-18, the composition may include caspase-1 inhibitors, antibodies against IL-18, antibodies against any of the IL-18 receptor subunits, inhibitors of the IL-18 signaling pathway, antagonists of IL-18. that compete with IL-18 and block the receptor of IL-18 and IL-18 binding proteins, isoforms, muteins, recombinant proteins, functional derivatives, active fractions or their cyclic permuted derivatives having the same activity.

IL-18BP and its isoforms, muteins, recombinant proteins, functional derivatives, active fractions or cyclic permuted derivatives, as described above, are the preferred active components of the pharmaceutical compositions.

Preferably, the interferon included in the pharmaceutical composition is IFN-β.

In another preferred embodiment, the pharmaceutical composition comprises therapeutically effective amounts of an IL-18 inhibitor, optionally an interferon and a TNF antagonist. TNF antagonists may be antibodies that neutralize TNF activity, or soluble truncated TNF receptor fragments, also called TBPI and TPVP. The pharmaceutical composition of the invention may further comprise one or more COX inhibitors, preferably COX-2 inhibitors. The pharmaceutical composition of the invention may further include a thromboxane inhibitor such as aspirin and / or a lipid-lowering component, for example as articles.

The term "pharmaceutically acceptable" means that it includes any carrier which does not affect the biological activity of the active ingredient and which is not toxic to the host to which it is administered. For example, for parenteral administration, the active protein (s) may be formulated as a single dosage form for injection in a carrier, such as saline, dextrose solution, serum albumin, and Ringer's solution.

The active ingredients of the pharmaceutical composition according to the invention can be administered to the individual in various ways. Administration routes include intradermal, transdermal (eg, as slow-release agents), intramuscular, intraperitoneal, intravenous, subcutaneous, oral, epidural, topical and intranasal. Any therapeutically effective route of administration, for example absorption through epithelial or endothelial tissue, or through gene therapy, where the patient is administered a DNA molecule encoding the active ingredient (e.g., vector) that results in expression and secretion in vivo of the active component. In addition, the protein (s) of the invention may be administered together with other components of biologically active substances, such as pharmaceutically acceptable surfactants, excipients, carriers, diluents and carriers.

For parenteral administration (e.g., intravenously, subcutaneously, intramuscularly), the active protein (s) may be formulated as a solution, suspension, emulsion or lyophilized powder coupled to a pharmaceutically acceptable parenteral carrier (e.g., water, saline, dextrose solution). and additives that maintain isotonicity (eg mannitol) or chemical stability (eg preservatives and buffers). The preparation is sterilized by standard techniques.

The bioavailability of the active protein (s) according to the invention can also be improved by using conjugation methods that increase the half-life of the molecule in the human body, such as binding of the molecule to polyethylene glycol, as described in PCT Patent Application WO 92 / 13095.

The therapeutically effective amounts of the active protein (s) will be a function of many variables including the type of antagonist, the affinity of the antagonist for IL-18, any residual cytotoxic activity that the antagonist has, the route of administration, the clinical condition of the patient (including desire for the patient) maintenance of non-toxic level of endogenous IL-18 activity).

A therapeutically effective amount is that amount which, when administered, the IL-18 inhibitor results in inhibition of the biological activity of IL-18. The individual dose administered as a single or multiple dose will vary depending on various factors including the pharmacokinetic properties of the IL18 inhibitor, routes of administration, patient status and characteristics (gender, age, body weight, health, judgment), grade symptoms, concurrent treatments, frequency of treatment and desired effect. Adjustment and manipulation of established dose ranges are well known to those skilled in the art as well as in vitro and in vivo methods for determining the inhibition of IL-18 in an individual.

According to the invention, the IL-18 inhibitor is used in an amount of about 0.0001 to 10 mg / kg or about 0.01 to 5 mg / kg body weight, or about 0.01 to 5 mg / kg body weight, or about 0.1 to 3 mg / kg body weight, or about 1 to 2 mg / kg body weight. Other preferred amounts of IL-18 inhibitors are amounts of about 0.1 to 1000 pg / kg body weight, or about 1 to 100 pg / kg body weight, or about 10 to 50 pg / kg body weight.

The method of administration which is preferred according to the invention is subcutaneous administration. Another preferred route of the invention is intramuscular administration.

In other preferred embodiments, the IL-18 inhibitor is administered daily or every other day.

Daily doses are usually given as two separate doses or in the form of sustained-release effective doses to obtain the desired results. Second or subsequent applications may be made at the same dose less than or greater than the original or previous dose administered to the individual. A second or subsequent application may be made during or before the onset of the disease.

According to the invention, the IL-18 inhibitor may be administered to the patient prophylactically or therapeutically, before, concurrently or sequentially with other therapeutic regimens or preparations (e.g., multi-drug regimens), in a therapeutically effective amount. The active ingredients, which are co-administered with other therapeutic agents, can be administered with the same or different compositions.

The invention further relates to a method of treating and / or preventing atherosclerosis, comprising administering to a host in need thereof an effective inhibitory amount of an IL-18 inhibitor.

The invention further relates to a method of preventing and / or treating atherosclerosis comprising administering to a host in need thereof an expression vector comprising the coding sequence of the IL-18 inhibitor.

In a preferred embodiment of the invention, the expression vector is administered continuously, and more preferably by intramuscular injection.

The invention further relates to the use of IL-18 as a diagnostic marker for poor clinical prognosis in heart failure. Poor clinical prognosis involves any worsening of the patient's condition, such as recurrent conditions, or even death, followed by myocardial infarction.

It is preferable to use IL-18 as a diagnostic marker for recurrent conditions after the first case of heart failure. Periodic conditions include, but are not limited to, recurrent ischemia, re-vascularization, progressive atherosclerosis, or re-hospitalization for heart failure.

Having described the invention, it will be much better understood by the following examples which enable it to be illustrated and in no way intended to limit the present invention.

EXAMPLES FOR THE IMPLEMENTATION OF THE INVENTION

Materials and methods

Samples

Forty-one human atherosclerotic plaques were obtained from 36 patients undergoing carotid endarterectomy. As controls, 2 carotid and 3 internal mammary arteries were obtained, without atherosclerosis (2 with minimal fibromuscular seals), at autopsy, or during surgical coronary bypass surgery. They were immediately placed in liquid nitrogen and stored at -80 ° C. The plaques used for protein and RNA extraction were rapidly rinsed and placed in liquid nitrogen before being stored at -80 ° C. For immunohistochemical studies, the plates are placed in fresh 4% paraformaldehyde for 2 hours, then transferred into 30% sucrose-PBS solution before freezing and breaking at the optimum cutting temperature in the required medium (OSC Compound , Miles Inc, Diagnostics Division) with liquid nitrogen and stored at -80 ° C for cryostat sections. Several sections of 8- to 10-µm thickness were obtained from each sample for histological analysis and immunohistochemical studies.

Classification of patients

To investigate the possible relationship between IL-18 / IL-18BP expression and signs of plaque instability, we collected, in a suspicious and random manner, clinical data from 23 consecutive patients (out of 36) undergoing endarterectomy between May and August 2000. Information on the presence or absence of intra-plaque ulcers in macroscopic examination is submitted on a permanent basis by the surgeon who performs the endarterectomy procedure. This allows the plates to be classified as ulcers with or without ulcers. In addition, patients were classified according to clinical symptoms into two separate groups. Patients who have clinical symptoms of cerebral ischemic attack related to carotid stenosis are classified as symptomatic. Endarterectomy is performed 2-66 days (17.6 ± 5.3 days) after the onset of clinical symptoms in these patients. Patients who have never had symptoms of cerebral ischemia in the carotid artery are classified as asymptomatic. Asymptomatic carotid stenoses are detected on the basis of systematic clinical studies of patients with coronary or peripheral disease, the severity of the disease being determined using periodic Doppler ultrasound by an experienced, certified ultrasound examiner. Although asymptomatic patients have never had a case of ischemia in the area of carotid stenosis, carotid endarterectomy has been shown to be favorable for these patients, according to researchers (28) in the Asymptomatic Carotid Atherosclerotic Study (ACAS).

Western blot analysis

Proteins from 12 atherosclerotic plaques and 5 control normal arteries were extracted. The frozen samples were destroyed under liquid nitrogen. The powder samples were resuspended in ice-cold lysis buffer [20 mmol / L Tris-HCl, pH 7.5, 5 mmol / L EGTA, 150 mmol / L NaCl, 20 mmol / L glycerophosphate, 10 mmol / L NaF, 1 mmol / L sodium orthovanadate, 1% Triton X-100, 0.1% Tween 20, 1 pg / mL aprotinin, 1 mmol / L PMSF, 0.5 mmol / L N-tosyl-TCphenylalanine chloromethyl ketone (TPCK), 0.5 mmol / L AND (a) -p-tosylL-lysine chloromethyl ketone (TLCK)] at a ratio of 0.3 mL / 10 mg wet weight. The extracts were incubated on ice for 15 minutes and then centrifuged (12,000 g, 15 minutes, 4 ° C). Detergent soluble supernatant fractions were maintained and protein concentrations in the samples were equilibrated using Bio-Rad protein assay.

For Western blot analysis for IL-18 and IL-18R, protein extracts were boiled for 5 minutes and applied to 7.5% or 15% SDS-polyacrylamide gel. IL-18BP, rhIL-18 purified from E.Coli (Serono Pharmaceutical Research C Institute, Geneva), was bound to Affligel 15 (Biorad) with 1 mg / ml of resin according to the manufacturers protocol. Protein extracts (60 pg) were incubated overnight at 4 ° C on a roller with 20 μΐ of resin adjusted to 500 μΐ was PBS 0.05% Tween. To remove any non-specific binding, the resin was centrifuged and washed with 10 mM Tris pH 8, 140 mM NaCl, 0.5% Triton-X-100 (Fluka), 0.5% deoxycholate, then 50 mM Tris pH 8,200 mM NaCl, 0.05% TX100, 0.05% .Nonidate P40 (Fluka), 2 mM CHAPS (Boehringer, Mannheim), followed by a final wash with 50 mM Tris, pH 8. The resin was then centrifuged, resuspended in the sample buffer. under reducing conditions, it was boiled for 5 minutes and finally applied to a 10% SDS polyacrylamide NuPAGE gel (Invitrogen).

Samples were electrophoretically transferred from the polyacrylamide gel to nitrocellulose. The nitrocellulose membranes were saturated for 2 hours at room temperature in TBST [50 mmol / L Tris-HCl (pH 7.5), 250 mmol / L NaCl, and 0.1% Tween saline] containing 5% skimmed milk powder. The membranes were then incubated with goat anti-human IL-18 and IL-18R (α-chain) polyclonal antibodies (1 pg / ml) (R&D Systems), a murine anti-human IL-18 BP monoclonal antibody (Mab 657.27 with 5 pg / ml) (Corbaz et al., 2000 article presented), rabbit anti-human caspase-1 polyclonal antibody (1 pg / ml) (A-19, Santa Cruz). The specificity of Mab 657.27 was analyzed on a cut membrane strip using a competitor using 200 x molar excess of rhIL-18BP-6his (purified from Chinese hamster ovary cells, Serono Pharmaceutical Research Institute), coincubated with Mab 657.27 at 5 pg / ml. , for 1 hour. After incubation with the appropriate HRP conjugated antibodies, chemiluminescent substrates (ECL, Western blotting; Amersham Corp) were used to show the positive bands according to the manufacturer's instructions, and the bands were visualized after exposure with Hyperfilm ECL (Amersham Corp).

Immunohistochemistry

Frozen sections of 6 atherosclerotic plaques were incubated with 1:10 normal horse serum or 1:10 normal goat serum for 30 minutes at room temperature, washed once with PBS, then incubated or with the first mouse monoclonal antibody against CD68 for macrophage identification (DAKO-CD68, KR1), or with the first murine monoclonal antibody against human smooth muscle α-actin (1A4, DAKO), to identify smooth muscle cells. Specific goat polyclonal antibodies (R&D Systems) were used to identify the IL-18 and IL-18 receptors in atherosclerotic plaques at a dilution of 5 pg / mL. IL-18 BP was detected using a specific monoclonal antibody directed against recombinant human IL-18BP isoform (H2O) (Corbaz et al., 2000 article presented). After washing with PBS, the sections were incubated with the following second biotinylated antibodies: biotinylated equine anti-mouse IgG (Vector Laboratories, Inc), at a dilution of 1: 200, to detect staining with antibodies against CD68, smooth muscle α-actin and IL- 18 BP, and biotinylated equine anti-goat IgG (Vector), at a dilution of 1: 200, for detection of anti-1E-18 and αHTH-IL-18 receptor antibodies. Immunoblotting was visualized using avidinbiotin HRP imaging systems (Vectastain ABC kit PK-6100 Vector). Adjacent slices with isotype-like unbound antibodies were stained as negative controls instead of primary antibodies.

RNA preparation

From 29 atherosclerotic plaques, total RNA was extracted into acid guanidine thiocyanate solution and extracted with phenol and chloroform according to the method of Chomczynski and Sacchi (29). The purified RNA was dissolved in water and the concentration and measured by absorbance at 260 nm. RNA integrity was assessed by electrophoresis on a 1% agarose gel. The cDNA was synthesized from pg total PHK using the Promega reverse transcriptase system according to the manufacturer's protocol.

Semi-quantitative and Real-time PCR of human IL-18 and IL18BP in human atherosclerotic plaques

Semi-quantitative PCR reactions were performed in a total volume of 50 µl in the presence of 1 H of AmpliTaq DNA Polymerase (Perkin Elmer, Roche, U.S.A), 2.5 mM dNTPs (Amersham, U.S.A) and 50 pmoles of forward and reverse PCR primers. The reactions were incubated in PTC200 Peltier Effect Thermal Cycler (MJ Research, U.S.A) under the following conditions: denaturation for 1 minute at 94 ° C, hybridization for 1 minute at 55 ° C, and continued reaction for 1 minute at 72 ° C. To ensure comparison of the amount of PCR products during the linear phase of the PCR reaction, IL-18BP, IL-18 and β-actin were analyzed after 25, 28 and 31 cycles. The optimum number of cycles for IL-18BP, IL-18 and β-actin was determined before saturation of the bands (31, 28 and 25, respectively). PCR primers were constructed based on published sequences (AF110799, D49950, X00351) as follows:

IL-18 reverse 5′-GCGTCACTACACTCAGCTAA-3 ′;

right

IL18BP, right reverse β-actin, reverse

5'-GCCTAGAGGTATGGCTGTAA-3 ';

'- ACCTGTCTACCTGGAGTGAA-3';

'-GC ACGA AGATAGGAAGTCTG-3';

'-GGAGGAGCAATGATCTTGATCTTC-3';

rights 5 '-GCTCACCATGGATGATGATATCGC-3'.

In order to exclude amplification of possible genomic DNA contaminating the samples, PCR reactions were performed in the absence of a cDNA template. PCR products (10µ1) were analyzed on 1% agarose gels by electrophoresis in 1X TAE buffer. The size of

PCR products were checked by comparison with a 1 kb ladder (Gibco) after staining of the calves. Estimation of the relative amount of ethidium bromide strips under UV light is performed using Kodak Digital Sciences analytical software and reported as the ratio of the target gene (hIL-18BP, hIL-18) to the housekeeping gene (hp-actin) .

SYBR Green Real Time PCR primers were designed for IL18, IL-18BP and GAPDH (house-keeping control) using Primer Express software from PE Biosystems according to published sequences (AF110799, D49950, NM 002046) as follows: IL-18 , reverse 5'-CAGCCGCTTTAGCAGCCA-3 ';

rights 5 '-CAAGGAATTGTCTCCCAGTGC-3';

IL18BP, reverse 5′-AACCAGGCTTGAGCGTTCC-3 ′; rights 5 '-TCCCATGTCTCTGCTCATTTAGTC-3';

GAPDH, reverse 5′-GATGGGATTTCCATTGATGACA-3 ′; rights 5 '-CC ASCC ATGGC AAATTS-3';

intron-GAPDH, reverse 5 '-CCTAGTCCCAGGGCTTTGATT-3'; rights 5 '-CTGTGCTCCCACTCCTGATTTC-3'.

Specificity and optimal primer concentration are analyzed. Possible genomic DNA contamination is ruled out by conducting the PCR reaction with specific intronGAPDH primers. The absence of non-specific amplification is confirmed by electrophoretic analysis of PCR products with

3.5% agarose gel. SYBR Green Real-Time PCR was performed with 5 μΐ / well of RT products (0.5 ng total RNA), 25 μΐ / well of SYBR Green PCR master mix (PE Biosystem, CA, USA), with AmpErase Uracil N-Glycosylase ( UNG) (0.5 Unit / well) and 20 μΐ primers (300 nM). PCR was performed at 50 ° C for 2 minutes (for AmpErase UNG incubation to remove any uracil included in the cDNA),

95 ° C for 10 min (for AmpliTaq Gold activation) and then performing 40 cycles at 95 ° C for 15 seconds, 60 ° C for 1 minute with the ABI PRISM 7700 Detection System. In this way, cDNA samples are amplified by reverse transcriptase and their Ct (threshold cycle) values determined. All Ct values were normalized to the housekeeping gene GAPDH. Single specific DNA strand for IL-18, IL-18BP and GAPDH was observed using gel electrophoretic analysis.

The principle of real-time detection using the "SYBR Green PCR master mix" is based on the direct detection of the PCR product by measuring the increased fluorescence caused by the binding of SYBR Green dye to double stranded DNA.

Statistical analysis

Data are given as mean ± SEM. IL-18 levels were compared between groups using the Mann-Whitney test. A value of p 0.05 was considered statistically significant.

Example 1: Oxidative Lipoprotein Induced Inhibitors of IL-18 Inhibitors of Endothelial Cell Death (oxLDL)

Cultured human umbilical vein endothelial cells (HUVECs) were exposed for 16 hours to oxLDL, in the presence or absence of an IL-18 binding protein or anti-IL-18 antibody. As shown in Figure 1, 83% of HUVECs die after exposure to oxLDL. Co-incubation with IL18BP or anti-IL-18 antibody rescues the cells almost completely from death. No cell death was observed using IL18BP. 89% of cells survive when an anti-IL-18 antibody is used.

This experiment clearly demonstrates the protective effect of two different IL-18 inhibitors against cell death due to apoptosis in atherosclerotic plaque.

Example 2: Expression of IL-18 Protein and its Endogenous IL-18 BP Inhibitor in Atherosclerotic Plates

Western blot analyzes of protein extracts from 12 carotid atherosclerotic arteries and 5 normal controls were performed. The active-form protein IL-18 is strongly expressed in all atherosclerotic plaques until almost no expression is detected or it is completely missing in normal arteries (Figure 2). Starts 1 to 4 contain samples of atherosclerotic plaques, starts 5 to 7 are from normal arteries. Interestingly, the detection of the active form of IL-18 probably correlates with the expression of the active form of caspase-1, which is involved in the processing of IL-18 (Figure 2, fourth row). Significant expression of the IL-18 receptor protein (osverig) was also observed in all atherosclerotic plaques, compared to the very low expression level in normal arteries (second row Figure 2). In addition, most of the atherosclerotic plaques express IL-18BP, although the expression level is heterogeneous (Figure 2 first row).

Example 3: Cellular localization of the IL-18 protein and its endogenous IL-18BP inhibitor in atherosclerotic plaques

To determine the cellular localization of IL-18 and IL-18BP, immunohistochemical studies were performed on 6 carotid atherosclerotic plaques. As can be seen from Figure 2, IL-18 is mainly expressed in macrophages, these cells being probably the major source of IL-18 in the plaque (not shown). These areas are also rich in CD3-positive lymphocytes. However, T lymphocytes do not appear to be directly involved in the production of IL-18. IL-18 is also expressed in some internal smooth muscle cells and less frequently in endothelial cells. In contrast, significant expression of IL-18BP is detected in capillary endothelial cells is plaque and in those of the lumen surface, although expression is not detected in all vessels. Relatively lower and more heterogeneous expression of IL-18 BP is also detected, mainly extracellularly, in some macrophage-rich regions.

Example 4: Expression of IL-18 and IL-18BP mRNA transcripts in atherosclerotic plaques and link is plaque instability

To determine whether human IL-18 and IL-18BP mRNA were expressed in human carotid atherosclerotic plaques, semi-quantitative RT-PCR was performed with six atherosclerotic plaques (Figure 3). IL-18 and IL-18BP mRNAs are detected in all atherosclerotic plaques, although the amount of mRNA for IL18 and IL-18BP is heterogeneous. Therefore, to accurately calculate the levels of IL-18 and IL-18BP mRNA expression, 23 atherosclerotic plaques were further analyzed using the SYBR Green Real-Time PCR method (Figure 4). The plaques are characterized by clinical and pathological examination as symptomatic (unstable) or asymptomatic (stable) plaques containing macroscopically or not containing ulcers. The clinical characteristics of the patients are summarized in Table 4. The percentage reduction in carotid diameter (60% - 95%) and risk factors including age, diabetes, hypercholesterolemia, hypertension and smoking did not differ between the two groups.

Table 4

Characteristics of the patient

Asymptomatic Symptomatic Patients (n = 9) Patients ¹ (n = 14) Age 66.9 ± 4.0 70.2 ± 3.9 Genus Male (8) Men's (9) Hypertension ² 8 9 Hypercholesterolemia ³ 4 8 Diabetes 3 1 Currently smoking 7 8 Coronary artery disease 5 4 'These patients are with temporary or persistent ischemic

cerebral attack 2-66 days before endarterectomy.

² Number of patients with clinical hypertension treated with antihypertensive components.

³ Number of patients with clinical hypercholesterolemia who have been treated with lipid-lowering drugs.

The amount of IL-18 was found to be up-regulated in symptomatic versus asymptomatic atherosclerotic plaques (2.03 ± 0.5 vs. 0.67 ± 0.17, respectively) (Figure 4A). Statistical analysis showed that this increase in IL-18 production observed in symptomatic plaques was of high significance (p <0.0074), whereas the increased amount of IL-18BP observed in symptomatic versus asymptomatic plaques was not (4.64 ± 0.98 vs. 2.5 ± 0.92, respectively) (Figure 4B). In other words, although both symptomatic and asymptomatic groups showed a positive correlation between IL-18 and IL-18BP mRNA, the slopes were significantly different between the two groups (symptomatic group: slope 1.16 [0.19-2.14], d = 0.36 versus asymptomatic group : slope 4.79 [2.39-7.20], r ² = 0.76; p <0.05). Therefore, it seems that the relative increase in IL-18BP expression in the symptomatic group is not sufficient to compensate for the increased expression of IL-18. Furthermore, since the presence of inflammation is considered as a distinguishing feature of plaque instability, a statistical analysis of plaques with or without intra-plaque inflammation was further performed and a marked upregulation of IL-18 in plaques was demonstrated. ulcers (p <0.018) were present (Figure 4C).

These data indicate that the increase in IL18 expression observed in atherosclerotic plaques correlates with plaque instability.

Example 5: IL-18BP modulates atherosclerotic lesion development and stability in in vivo disease models

Methods

Characteristics of the patient

Plasma samples are obtained from patients with acute ischemic coronary syndromes (unstable angina and myocardial infarction) less than 7 days after the onset of symptoms. Unstable angina is defined as associated with typical chest pain, or with ischemic changes in the electrocardiogram, or with the presence of coronary artery disease. Myocardial infarction is diagnosed on the basis of typical ischemic changes in the electrocardiogram associated with a significant increase in myocardial enzymes (creatine phosphokinase and troponin I) in the circulating blood. Patients without ischemia are recruited to the same cardiology department and are completely deprived of ischemia signals. Plasma levels of human IL-18 were determined using a commercially available kit (MBL, Japan).

In vivo intramuscular electrotransfer of mouse IL-18BP expression plasmid

Fourteen male C57BL / 6 apoE KO mice, 14 weeks of age, received, at intervals of three weeks, 3 injections of the expression IL-18BP plasmid, pcDNA3-IL18BP. Control mice (n = 19) were injected with a control blank plasmid. The mouse IL-18BP isoform d cDNA isolated as described (accession number # Q9ZOM9) (23) was subcloned into EcoR1 / Not1 sites from an expression vector of a mammalian pcDNA3 cell, under the control of a cytomegalovirus promoter (Invitrogen). The construct termed 334.yh is shown in Figure 5. Similarly, a control plasmid devoid of therapeutic cDNA was constructed. IL-18BP or the control expression plasmid (60 pg) was injected into both the tibial and cranial muscles of the anesthetized mouse as previously described (13). Briefly, electrical impulses through the skin (8 rectangular (square) electrical impulses 200 V / cm, 20 msec duration, 2 Hz) are supplied by a PS-15 electropulsor (Genetronics, France) using two stainless steel flat electrodes mounted 4.2 to 5.3 mm from each other on each side of the foot.

Elisa mIL-18BP

The plates were coated overnight with r-mI-18BP (1-affinity purified rabbit polyclonal antibody (5 pg / well). The soluble mIL-18BP was detected using biotinylated rabbit polyclonal antibody (0.3 pg / ml) prepared against E. coli r-mIL-18BP (Peprotec) followed by extravidine peroxidase (1/1000) (Sigma) The bound rabbit polyclonal antibody was analyzed by Western blot to confirm the specificity of mIL-18BP. recombinant mIL-18BPd derived from HEK 293 cells The sensitivity of ELISA is 5 ng / ml.

Mouse analysis

Cryostatic sections (8 pm) were obtained from the aortic sinus and used for the detection of lipid deposition using Oil red, for collagen detection using Sirius red, and for immunohistochemical analysis as described previously (13). The sections were stained with specific first antibodies: anti-mouse macrophage, clone MOMA2 (BioSource), conjugated alkaline phosphatase anti-α-actin for smooth muscle cells and anti-CD3 for

T lymphocytes (Dako) as previously described (13). The use of the TUNEL technique (13) has been used to detect cell death. CD3 positive cells were counted microscopically by chance. Atherosclerotic plaques in the aortic sinus and areas stained positive for macrophages, smooth muscle cells, collagen or TUNEL were measured using a computer generated quantitative image assay (NS15000, Microvision) as previously described (13). Staining with non-immune isotype-like immunoglobulins determines the specificity of the immunostaining. The specificity of TUNEL is determined by the release of the terminal deoxynucleotidyl transferase enzyme. Thoracic aortas covering the left subclavian artery to the renal arteries were fixed with 10% buffered formalin and stained for lipid deposition with Oil red. They are then opened lengthwise and the percentage of lipid deposition calculated using a computer generated image quantification image (NS15000, Microvision).

Results

This study analyzes the hypothesis that regulation of IL-18 / IL-18BP plays a critical role for both atherosclerosis and plaque stability. Plasma IL-18 levels were measured in patients with acute coronary syndromes (30 men, 18 women, mean age 66.2 ± 1.8 years, of whom 14 had unstable angina and 34 had myocardial infarction) and in control patients without ischemia, recruited from the same cardiology department (10 males, 3 females, mean age 60.0 ± 5.2 years). Plasma IL-18 levels increased significantly in patients with acute coronary disease compared to controls (146.9 ± 17.1 vs. 73.0 ± 12.2 pg / ml, respectively, p <0.05) compared to circulating IL-18BP levels, which were slightly increased (20.1 ± 2.7 versus 7.5 ± 2.5 ng / ml, respectively, p = 0.06). In addition, IL-18 levels correlate with the severity of the disease, with the highest levels observed in patients with severe ischemic heart dysfunction and clinical reports of pulmonary edema (224.03 ± 39.1 pg / ml, p <0.001 compared to controls) . These results obtained from patients with acute heart disease, together with previous observations that IL-18 is increased in atherosclerotic plaques in stroke patients [Mallat, 2001], suggest a possible important role for the regulation of IL-18 / IL-18BP in atherosclerotic process.

Therefore, we tested this hypothesis using apoE knockout (KO) mice that develop spontaneously similar to human, atherosclerotic lesions. Fourteen mice, 14 weeks of age, received in addition IL-18BP intramuscularly, by in vivo electrotransfer of plasmid DNA encoding murine IL-18BPd, while 19 controls of similar age received an empty plasmid. Plasmid electrotransfer was repeated every 3 weeks and mice were killed 23 weeks at 9 weeks post-treatment. Mouse IL-18BP plasma levels were lower than the limit (5 ng / ml) in apoE KO mice injected with the empty plasmid. However, a single injection of IL-18BP plasmid resulted in high levels of IL-18BP in the blood, with a maximum increase of 2 days after injection (323.5 ± 100.9 ng / ml) and 127.4 ± 35.4 ng / ml measured after 2 weeks. Nine weeks after treatment with either IL-18BP or blank plasmid, total cholesterol (489.4 ± 34.6 vs. 480.8 ± 36.3 mg / dl, respectively) and serum levels of high-density lipoproteins (52.3 ± 9.4 vs. 48.8 ± 5.1 mg / dl , respectively) are no different between the two groups. A moderate but significant increase in animal weight was observed in the IL-18BP treated group compared to the control group (31.8 ± 0.9 versus 28.6 ± 0.8 g, respectively, p <0.05).

The result of the addition of IL-18BP in atherosclerosis was evaluated in two different sections: the descending thoracic aorta and the aortic sinus. The thoracic aorta was selected to determine the role of IL-18BP in the development of fatty compaction (atherogenesis), since thoracic atherosclerotic lesions were absent almost completely at 14 weeks of age (data not shown) where IL-18BP transfection began. The aortic sinus, where atherosclerotic lesions are already present at 14 weeks of age (data not shown), is examined for advanced plaque progression and composition, an important factor for plaque stability. IL-18BP treatment of apoE KO mice significantly affects the development and progression of the atherosclerotic lesion. Thoracic aorta examination showed a significant reduction in lipid deposition in mice treated with the IL-18BP plasmid compared to the empty plasmid (Figure 6). A computer-generated quantitative analysis image showed a 69% reduction in the degree of atherosclerotic lesions (p <0.0001) (Figure 6), indicating the critical, decisive role of IL-18 in atherosclerosis. In addition, treatment with the IL-18BP plasmid for only 9 weeks significantly reduced the development of advanced atherosclerotic plaques in the aortic sinus (24% reduction in plaque size, p = 0.01) compared to the treatment with the empty plasmid (Figure 7).

Much more importantly, the composition of advanced lesions, as a major factor in plaque instability, is strongly influenced by IL-18BP treatment. Atherosclerotic lesions of mice treated with the IL-18BP plasmid showed a significant 50% reduction in macrophage infiltration (p <0.0001) (Figure 8) containing 67% less T lymphocytes (p <0.005) (Figure 8) increased accumulation in smooth muscle cells (p <0.05) (Figure 9). In addition, these significant changes in the damaged cellular composition were associated with a significant 85% increase in collagen content (p <0.0005), which was determined by staining with Sirius red and a decrease in total lipid content.

Therefore, treatment with IL-18BP leads to a significant reduction in the inflammatory process in atherosclerotic lesions and induces a healing process, a characteristic of stable atherosclerotic plaques. In addition, the marked reduction in the inflammatory component of lesions in IL18BP-treated mice was associated with a significant reduction in cell death in plaque D9 ± 0.9% in IL-18BP treated mice relative to

10.5 ± 3.6% of controls, p <0.05), thereby reducing the enlargement and thrombogenicity of the acellular core [Mallat, 1999].

Conclusions:

Using a well established murine model resembling atherosclerosis: human, the results reported above clearly demonstrate the unexpected and critical role of regulation of IL-18 and IL-18BP in the development, progression and stability of atherosclerotic plaques. As well as preventing the formation of a prn lesion in the thoracic aorta, inhibition of IL-18 activity by the addition of IL-18BP also strongly affects the composition of advanced lesions in the aortic sinus, inducing a switch to a stable plaque phenotype.

Clinical prognosis in patients with atherosclerosis depends only partially on the size of the lesions. The idea that quality (the composition of the plaque) rather than the size of the lesion may be an even better prophet for the incidence of ischemic cases is increasingly accepted today. Indeed, the severe clinical manifestations of atherosclerosis (heart and brain infarctions) are mainly due to obstruction of the vascular lumen by a thrombus that forms at the site of contact of the destroyed atherosclerotic plaque (19). Pathologic studies have shown that vulnerable or unstable plaques that are prone to destruction or destroyed are rich in inflammatory cells and show significant loss of smooth muscle cells and contained in collagen (20, 21). In addition, such plaques show a significant increase in apoptotic cell death, leading to the formation of a highly thrombogenic lipid core (13, 22). It is noteworthy that all of these plaque instability signals decrease significantly in IL-18BP-treated mice, indicating that IL-18 signaling is a major factor in plaque instability.

The practical relevance of the results obtained in apoE KO mice with respect to human disease is enhanced by our findings for increased levels of circulating IL-18 in patients with acute coronary syndromes and increased production of IL-18 in unstable carotid atherosclerotic plaques, responsible for stroke. Taken together, these findings identify IL-18 activity inhibitors as novel important therapeutics for preventing and treating the development of atherosclerotic plaques and limiting complications in their development.

Example 6: Elevated IL-18 levels correlate with recurrent cases of patients with heart failure

IL-18 levels are measured in the blood serum of patients by ELSIA with an IL-18 specific antibody.

All 56 ischemically ill patients, as well as patients without ischemia, heart failure or patients who do not have one, are analyzed.

In patients who died later, IL-18 levels were 216.0 + 41.5 pg / ml, compared to 112.2 + 12.2 pg / ml in non-lethal patients (p = 0.0018).

In patients who experience any recurrent cases, such as death, recurrent ischemia, revascularization, progressive atherosclerosis, or re-hospitalization for heart failure, the following IL-18 levels were measured: 165.8 + 23.8 versus 107.7 + 14.6, at patients who did not experience recurrent cases (p = 0.03).

These results indicate that IL-18 levels are significantly elevated in patients with poor clinical prognosis, such as those with recurrent conditions or even in those who die.

IL-18 levels were measured in the blood samples of 16 patients who did not suffer from ischemic disease, who had or who lacked heart failure. In patients who die late, the levels are 199.0 + 34.8 pg / ml versus 95.3 + 20.4 pg / ml, in those patients who remain alive (p = 0.09).

Patients with any recurrent conditions: 146.6 + 34.4 pg / ml IL-18 versus 95.4 + 23.9 pg / ml in patients who did not experience recurrent cases (p = 0.03). Although differences in IL-18 levels are not statistically significant, due to the small number of patients, there is a clear tendency for an increase in IL-18 levels.

In patients with ischemia, IL-18 levels were 214.2 + 45.9 pg / ml in patients who died, compared to 118.4 + 12.8 pg / ml in those who remained alive (p = 0.007).

In patients with any recurrent condition, the measured IL-18 levels were 162.8 + 24.7 pg / ml compared to 116.2 + 16.0 in patients who did not experience recurrent conditions.

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Claims (48)

Patent Claims Use of an IL-18 inhibitor for the manufacture of a medicament for the treatment and / or prevention of atherosclerosis. Use of an IL-18 inhibitor for the manufacture of a medicament for the treatment and / or prevention of thrombosis by atherosclerotic plaque. Use of an IL-18 inhibitor for the manufacture of a medicament for the treatment and / or prevention of ulcerative atherosclerotic plaque. 4. Use of an IL-18 inhibitor for the manufacture of a medicament for the treatment and / or prevention of atherosclerotic plaque destabilization. 5. Use of an IL-18 inhibitor for the manufacture of a medicament for the prevention and / or treatment of ischemic syndromes due to plaque instability. Use of an IL-18 inhibitor for the manufacture of a medicament for the treatment and / or prevention of atherosclerotic plaque destruction. Use of an IL-18 inhibitor for the manufacture of a medicament for the treatment and / or prevention of recurrent heart failure conditions. Use according to Claim 7, wherein the heart failure is ischemia. Use according to claim 7, wherein the heart failure is not ischemia. The use according to any one of claims 1 to 9, wherein the IL-18 inhibitor is selected from the group consisting of ICE inhibitors, antibodies against IL-18, antibodies against any of the receptor IL-18 subunits, IL-18 receptor pathway inhibitors, IL-18 antagonists that compete with IL-18 and block IL-18 receptor and IL-18 binding proteins, isoforms, muteins, recombinant proteins, functional derivatives, active fractions or cyclic permuted derivatives. The use of Claim 10, the IL-18 inhibitor is an antibody directed against IL-18. The use of Claim 11, the antibody is a human-like antibody. where where The use according to Claim, the antibody is a human antibody. Use according to Claim 11, where 10, wherein the inhibitor of the action of IL-18 is IL-18BP, or an isoform, mutein, derivative or fragment thereof. The use according to any one of Claims 10 to 14, wherein the IL-18 binding protein is PEOL. The use according to any one of claims 10 to 15, wherein the IL-18 inhibitor is a recombinant protein comprising all or part of an IL-18 binding protein bound to all or part of an immunoglobulin and wherein the recombinant protein binds with IL-18. The use of claim 16, wherein the recombinant protein comprises all or part of the immunoglobulin constant region. The use of claim 17, wherein the immunoglobulin is of the IgG1 or IgG2 isotype. The use according to any of the preceding claims, wherein the medicament further includes interferon, for simultaneous, sequential or self-administration. The use of claim 19, wherein the interferon is interferon-β. The use according to any of the preceding claims, wherein the medicament further includes a Tumor Necrosis Factor (TNF) antagonist for simultaneous, sequential or alone use. The use of claim 21, wherein the TNF antagonist is TBP I and / or TBP II. Use according to any of the preceding claims, wherein the medicament further includes a COX inhibitor for simultaneous, sequential or separate use. The use of claim 23, wherein the COXinhibitor is a COX-2 inhibitor. The use according to any of the preceding claims, wherein the medicament further includes a thromboxane inhibitor for simultaneous, sequential or alone use. The use of claim 25, wherein the thromboxane inhibitor is aspirin. The use according to any of the preceding claims, wherein the medicament further includes a lipid-lowering component for simultaneous, sequential or self-administration. The use of claim 27, wherein the lipid-lowering component is an HMG CoA inhibitor. The use of claim 28, wherein the HMG CoA inhibitor is articles. Use according to any of the preceding claims, wherein the medicament is used in combination with foods low in fat and / or low in cholesterol and / or low in salt. The use according to any of the preceding claims, wherein the IL-18 inhibitor is used in an amount of about 0.0001 to 10 mg / kg body weight or about 0.01 to 5 mg / kg body weight or about 0.1 to 3 mg / kg body weight, or about 1 to 2 mg / kg body weight. The use according to any of the preceding claims, wherein the IL-18 inhibitor is used in an amount of from about 0.1 to 1000 µg / kg body weight or about 1 to 100 µg / kg 0.1 to 3 mg / kg body weight, or about 10 to 50 gg / kg body weight. The use of any of the preceding Claims, wherein the IL-18 inhibitor is administered subcutaneously. The use of any of the preceding claims, wherein the IL-18 inhibitor is administered intramuscularly. The use of any of the preceding Claims, wherein the IL-18 inhibitor is administered daily. The use of any of the preceding claims, wherein the IL-18 inhibitor is administered daily. 37. Use of an expression vector comprising the coding sequence of an IL-18 inhibitor for the manufacture of a medicament for the treatment and / or prevention of atherosclerosis. The use of claim 37, wherein the expression vector is administered by electrotransfer. The use of claim 37 or 38, wherein the expression vector is administered systemically. Use according to any one of the preceding Claims, wherein the expression vector is administered intramuscularly. 41. Use of a vector for inducing and / or enhancing endogenous production of an IL-18 inhibitor in a cell, for the manufacture of a medicament for the treatment and / or prevention of atherosclerosis. 42. Use of a cell that is genetically modified to produce an IL-18 inhibitor for the manufacture of a medicament for the treatment and / or prevention of atherosclerosis. 43. A method of treating and / or preventing atherosclerosis, comprising administering to a host in need thereof an effective inhibitory amount of an IL-18 inhibitor. 44. A method of preventing and / or treating atherosclerosis, characterized in that it comprises administering to a host in need thereof an expression vector comprising the coding sequence of the IL-18 inhibitor. 45. The method of claim 44, wherein the expression vector is administered systemically. 46. The method of claim 44 or 45, wherein the expression vector is administered by intramuscular injection. 47. Use of IL-18 as a diagnostic marker for poor clinical prognosis in heart failure. 48. Use of IL-18 as a diagnostic marker, in recurrent conditions, after an initial case of heart failure. Patent Claims Use of an IL-18 inhibitor for the manufacture of a medicament for the treatment and / or prevention of atherosclerosis. Use of an IL-18 inhibitor for the manufacture of a medicament for the treatment and / or prevention of thrombosis by atherosclerotic plaque. Use of an IL-18 inhibitor for the manufacture of a medicament for the treatment and / or prevention of ulcerative atherosclerotic plaque. 4. Use of an IL-18 inhibitor for the manufacture of a medicament for the treatment and / or prevention of atherosclerotic plaque destabilization. 5. Use of an IL-18 inhibitor for the manufacture of a medicament for the prevention and / or treatment of ischemic syndromes due to plaque instability. Use of an IL-18 inhibitor for the manufacture of a medicament for the treatment and / or prevention of atherosclerotic plaque destruction. Use of an IL-18 inhibitor for the manufacture of a medicament for the treatment and / or prevention of recurrent heart failure conditions. Use according to Claim 7, wherein the heart failure is ischemia. Use according to claim 7, wherein the heart failure is not ischemia. The use according to any one of claims 1 to 9, wherein the IL-18 inhibitor is selected from the group consisting of ICE inhibitors, antibodies against IL-18, antibodies against any of the receptor IL-18 subunits, IL-18 receptor pathway inhibitors, IL-18 antagonists that compete with IL-18 and block IL-18 receptor and IL-18 binding proteins, isoforms, muteins, recombinant proteins, functional derivatives, active fractions or cyclic permuted derivatives. The use of Claim 10, the IL-18 inhibitor is an antibody directed against IL-18. where The use according to Claim, the antibody is a human-like antibody. The use according to Claim, the antibody is a human antibody. Use according to Claim 11, where 11, where 10, wherein the inhibitor of the action of IL-18 is IL-18BP, or an isoform, mutein, derivative or fragment thereof. The use according to any one of Claims 10 to 14, wherein the IL-18 binding protein is PEOL. The use according to any one of claims 10 to 15, wherein the IL-18 inhibitor is a recombinant protein comprising all or part of an IL-18 binding protein bound to all or part of an immunoglobulin and wherein the recombinant protein binds with IL-18. The use of claim 16, wherein the recombinant protein comprises all or part of the immunoglobulin constant region. The use of claim 17, wherein the immunoglobulin is of the IgG1 or IgG2 isotype. The use according to any of the preceding claims, wherein the medicament further includes interferon, for simultaneous, sequential or self-administration. The use of claim 19, wherein the interferon is interferon-β. The use according to any of the preceding claims, wherein the medicament further includes a Tumor Necrosis Factor (TNF) antagonist for simultaneous, sequential or alone use. The use of claim 21, wherein the TNF antagonist is TBP I and / or TBP II. Use according to any of the preceding claims, wherein the medicament further includes a COX inhibitor for simultaneous, sequential or separate use. The use of claim 23, wherein the COXinhibitor is a COX-2 inhibitor. The use according to any of the preceding claims, wherein the medicament further includes a thromboxane inhibitor for simultaneous, sequential or alone use. The use of claim 25, wherein the thromboxane inhibitor is aspirin. The use according to any of the preceding claims, wherein the medicament further includes a lipid-lowering component for simultaneous, sequential or self-administration. The use of claim 27, wherein the lipid-lowering component is an HMG CoA inhibitor. The use of claim 28, wherein the HMG CoA inhibitor is articles. Use according to any of the preceding claims, wherein the medicament is used in combination with foods low in fat and / or low in cholesterol and / or low in salt. The use according to any of the preceding claims, wherein the IL-18 inhibitor is used in an amount of about 0.0001 to 10 mg / kg body weight or about 0.01 to 5 mg / kg body weight or about 0.1 to 3 mg / kg body weight, or about 1 to 2 mg / kg body weight. The use according to any one of the preceding claims, wherein the IL-18 inhibitor is used in an amount of from about 0.1 to 1000 µg / kg body weight or about 1 to 100 pg / kg 0.1 to 3 mg / kg body weight, or about 10 to 50 pg / kg body weight. The use of any of the preceding Claims, wherein the IL-18 inhibitor is administered subcutaneously. The use of any of the preceding claims, wherein the IL-18 inhibitor is administered intramuscularly. The use of any of the preceding Claims, wherein the IL-18 inhibitor is administered daily. The use of any of the preceding claims, wherein the IL-18 inhibitor is administered daily. 37. Use of an expression vector comprising the coding sequence of an IL-18 inhibitor for the manufacture of a medicament for the treatment and / or prevention of atherosclerosis. The use of claim 37, wherein the expression vector is administered by electrotransfer. The use of claim 37 or 38, wherein the expression vector is administered systemically. Use according to any one of the preceding Claims, wherein the expression vector is administered intramuscularly. 41. Use of a vector for inducing and / or enhancing endogenous production of an IL-18 inhibitor in a cell, for the manufacture of a medicament for the treatment and / or prevention of atherosclerosis. 42. Use of a cell that is genetically modified to produce an IL-18 inhibitor for the manufacture of a medicament for the treatment and / or prevention of atherosclerosis. 43. A method of treating and / or preventing atherosclerosis, comprising administering to a host in need thereof an effective inhibitory amount of an IL-18 inhibitor. 44. A method for the prevention and / or treatment of atherosclerosis, V, comprising administering to a host in need thereof an expression vector comprising the coding sequence of an IL-18 inhibitor. 45. The method of claim 44, wherein the expression vector is administered systemically. 46. The method of claim 44 or 45, wherein the expression vector is administered by intramuscular injection. 47. Use of IL-18 as a diagnostic marker for poor clinical prognosis in heart failure. 48. Use of IL-18 as a diagnostic marker, in recurrent conditions, after an initial case of heart failure.